Bone Marrow Cultures Research Articles

Optically Controlled Living Micromotors for the Manipulation and Disruption of Biological Targets.

Bioinspired and biohybrid micromotors represent a revolution in microrobotic research and are playing an increasingly important role in biomedical applications. In particular, biological micromotors that are multifunctional and can perform complex tasks are in great demand. Here, we report living and multifunctional micromotors based on single cells (green microalgae: Chlamydomonas reinhardtii) that are controlled by optical force. The micromotor's locomotion can be carefully controlled in a variety of biological media including cell culture medium, saliva, human serum, plasma, blood, and bone marrow fluid. It exhibits the capabilities to perform multiple tasks, in particular, indirect manipulation of biological targets and disruption of biological aggregates including in vitro blood clots. These micromotors can also act as elements in reconfigurable motor arrays where they efficiently work collaboratively and synchronously. This work provides new possibilities for many in vitro biomedical applications including target manipulation, cargo delivery and release, and biological aggregate removal.

8. Modifiable polymer promotes a pro-osteogenic, M2-like macrophage phenotype and osteoblastic differentiation of progenitor cells

8. Modifiable polymer promotes a pro-osteogenic, M2-like macrophage phenotype and osteoblastic differentiation of progenitor cells

Kinetics of leukemic cells in 3D culture with stromal cells and with arginine deprivation stress

Previously, we established a three-dimensional (3D) bone marrow culture system that maintains normal hematopoiesis, including prolongation of hematopoietic stem cell proliferation and differentiation. To analyze the role of bone marrow stromal cells that compose the microenvironment, the growth of a leukemic cell line (K562) in the 3D condition and with arginine deprivation stress was compared with two-dimensional stromal cell monolayers (2D) and suspension cultures without stromal cells (stroma (-)). Arginine is essential for the proliferation and differentiation of erythrocytes. The proliferation and differentiation of K562cells cultured in the 3D system were stabilized compared with cells in 2D or stroma (-). Furthermore, the number of K562cells in the G0/G1 phase in 3D was increased significantly compared with cells grown in 2D or stroma (-). Interestingly, the mRNA expression of various hematopoietic growth factors of stromal cells in 3D was not different from 2D, even though supportive activity on K562cell growth was observed in the arginine deprivation condition. Thus, the hematopoietic microenvironment involves multi-dimensional and complex systems including biochemical and physiochemical factors that regulate quiescence, proliferation, activation, and differentiation of normal hematopoietic cells and cloned leukemic cells. Our 3D culture system may be a valuable new tool for investigating leukemic cell-stromal cell interactions invitro.

Portal venous repopulation of decellularised rat liver scaffolds with syngeneic bone marrow stem cells.

Liver transplantation is the only life-saving treatment for end-stage liver failure but is limited by the organ shortage and consequences of immunosuppression. Repopulation of decellularised scaffolds with recipient cells provides a theoretical solution, allowing reliable and timely organ sourcing without the need for immunosuppression. Recellularisation of the vasculature of decellularised liver scaffolds was investigated as an essential prerequisite to the survival of other parenchymal components. Liver decellularisation was carried out by portal vein perfusion using a detergent-based solution. Decellularised scaffolds were placed in a sterile perfusion apparatus consisting of a sealed organ chamber, functioning at 37°C in normal atmospheric conditions. The scaffold was perfused via portal vein with culture medium. A total of 107 primary cultured bone marrow stem cells, selected by plastic adherence, were infused into the scaffold, after which repopulated scaffolds were perfused for up to 30 days. The cultured stem cells were assessed for key marker expression using fluorescence-activated cell sorting (FACS), and recellularised scaffolds were analysed by light, electron and immunofluorescence microscopy. Stem cells were engrafted in portal, sinusoidal and hepatic vein compartments, with cell alignment reminiscent of endothelium. Cell surface marker expression altered following engraftment, from haematopoietic to endothelial phenotype, and engrafted cells expressed sinusoidal endothelial endocytic receptors (mannose, Fc and stabilin receptors). These results represent one step towards complete recellularisation of the liver vasculature and progress towards the objective of generating transplantable neo-organs.

Abstract 642: TRAF2 mediates sensitivity to immunomodulatory drugs via NF-κB and ERK signaling in myeloma cells

Abstract The development of novel agents including immunomodulatory drugs (IMiDs) lenalidomide (Len) and pomalidomide (Pom) has improved patient outcome in multiple myeloma (MM); however, acquired resistance to IMiDs commonly underlies relapse of disease. Previous studies show that IMiDs bind to the CRL4CRBN ubiquitin ligase cereblon (CRBN) and promote proteasomal degradation of IKZF1 and IKZF3 followed by downregulation of c-Myc and IRF4, resulting in MM cell growth inhibition. Importantly, some MM cells show resistance to IMiDs despite harboring high CRBN expression levels. Here we utilize genome-wide CRISPR-Cas9 knockout (KO) screening to identify novel mechanisms mediating resistance to IMiDs in MM. Among positively selected sgRNAs, we found three different sgRNAs targeting TRAF2 were enriched after IMiDs treatment. Individually KO of TRAF2 induced significant resistance of MM cells to Pom and Len treatment independent of CRBN-IKZF1/3 axis. TRAF2 KO cells show hyperactivation of the non-canonical NF-κB pathway and p52 KO re-sensitizes the cells to IMiDs treatment, suggesting that TRAF2 predominantly suppresses non-canonical NF-κB pathway, maintaining sensitivity to IMiDs in MM cells. We next examined the relevance of TRAF2 downregulation in the context of the bone marrow (BM) microenvironment. Co-culture of MM cells with either BM stromal cells (BMSCs) or culture supernatants (BM-CS) confers resistance to IMiDs. Importantly, TRAF2 in MM cells is downregulated by BMSCs and BM-CS co-culture. TNF-α was elevated in BM-CS, and exogenous TNF-α triggered decreased TRAF2 expression in MM cells, associated with resistance to IMiDs. Taken together, these results suggest that IMiDs resistance in the BM microenvironment may be mediated, at least in part, by TRAF2 downregulation. As observed in the TRAF2 KO, exogenous TNF-α hyper-activated the noncanonical NF-κB pathway without affecting CRBN expression, further confirming a major role of TNF-α mediating protection against IMiDs-driven cytotoxicity. Interestingly, we also found that TNF-α induces IMiDs resistance even in p52 KO cells, implicating additional mediating IMiDs resistance. Indeed, we found that ERK pathway was highly activated in TRAF2 KO MM cells, and confirmed that both BM-CS and TNF-α induced activation of ERK pathway in MM cells. Moreover, constitutive overexpression of phosphorylated-ERK-2 significantly reduced IMiDs-induced cytotoxicity. Finally, our in vitro and in vivo murine xenograft studies show that MEK inhibitor AZD6244 overcomes IMiDs resistance in TRAF2 KO MM cells or in BM-CS treated wild-type MM cells, indicating that MEK inhibitor can overcome IMiDs resistance in the BM microenvironment. Taken together, our studies therefore identify TRAF2 as a mediator of IMIDs sensitivity in MM cells independent of CRBN-IKZF1/3, and validate the therapeutic potential of MEK/ERK inhibitor to overcome IMiDs resistance. Citation Format: Jiye Liu, Teru Hideshima, Lijie Xing, Su Wang, Wenrong Zhou, Daisuke Ogiya, Kenneth Wen, Yu-Tzu Tai, Yong Cang, Kenneth C. Anderson. TRAF2 mediates sensitivity to immunomodulatory drugs via NF-κB and ERK signaling in myeloma cells [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 642.

Abstract 6199: Bone microenvironment-suppressed T cells increase osteoclast formation and breast cancer bone metastases

Abstract Bone metastases are a frequent complication in patients with advanced breast cancer and remain incurable. Thus, we need to explore new options for their treatment. Immunotherapy for the treatment of cancer is promising but remains untested for bone metastases. A potential limitation for such use is the ability of T cells to activate osteoclasts and bone resorption that releases pro-metastatic growth factors. Thus, we aim to characterize the effect of T cells in bone metastases. When inoculated in Balb/C SCID mice that lack T and B cells or in T cell-depleted Balb/C mice, 4T1 cells caused 72% and 38% less bone metastases, respectively (p<0.05), when compared to control mice. Histology confirmed that mice with a functional immune system have an increased number of osteoclasts at the tumor/bone interface. Consistently, the addition of mouse T cells to bone marrow culture ex vivo increased the formation of osteoclasts. However, this behavior was limited to non-activated T cells. Upon activation with antibodies against CD3 and CD28, T cells inhibited the formation of osteoclasts in a dose-dependent manner. This anti-osteoclastic effect was not due to a cytotoxic effect of T cells, and was consistent with decreased levels of the pro-osteoclastic Rankl and increased expression of anti-osteoclastic Ifng and Il4. When measured in vivo, Ifng was not detected, and levels of pro-osteoclastic Rankl and Tnfa were higher in T cells from 4T1 bone metastases compared to splenic T cells, suggesting that T cells in the bone marrow are not activated. Flow cytometry confirmed that only up to 15% of T cells in bone metastases expressed the activation marker CD69. Interestingly, T cells in the bone marrow could not be further activated ex vivo, unlike T cells from the spleen. This could be due to increased levels of the immunosuppressive monocytic (CD11b+Ly6C+Ly6G−) and polymorphonuclear (CD11b+Ly6G+Ly6C−) Myeloid Derived Suppressor Cells (MDSC) in bone metastases. To activate T cells in vivo, we attempted to target MDSCs using the PDE-5 inhibitor, Sildenafil, in combination with zoledronate, an FDA-approved bone resorption inhibitor. Still, there were no effects on MDSCs or the development of bone metastases. Therefore, we sought other therapeutic targets, such as immune checkpoints. Although only ~27% of T cells in 4T1 bone metastases were CTLA-4+, >70% of T cells were PD-1+, and ~80% of monocytic-MDSC were PD-L1+, which could trigger T cell suppression in bone. In conclusion, we found that the bone metastasis microenvironment suppresses T cells, making them pro-osteoclastic and increases the development of bone metastases. We also found that activation of T cells prevented the formation of osteoclasts. Therefore, the activation of T cells using immunotherapy, such as immune checkpoint inhibitors (anti-PD-1 or anti-PD-L1 antibodies), could be used for the treatment of patients with bone metastases. Citation Format: Danna L. Arellano, Juan A. Corral-Avila, Florian Drescher, Andrea Verdugo-Meza, Samanta Jimenez, Felipe Olvera-Rodriguez, Patricia Juarez, Pierrick Fournier. Bone microenvironment-suppressed T cells increase osteoclast formation and breast cancer bone metastases [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6199.

3056 – NES-EGFP TRANSGENIC MICE AS A MODEL FOR IN VIVO AND IN VITRO STUDY OF BONE-MARROW-DERIVED MESENCHYMAL STEM CELLS (MSCS)

Transgenic NES-EGFP mice developed by G. Enikolopov (Tg(Nes-EGFP)33Enik), which have a green fluorescent protein (EGFP) gene under the promoter of the nestin gene, had been previously used to show that early mesenchymal progenitors are in the population of CD45–GFP+ bone marrow (BM) cells. Here we show that NES-EGFP mice can serve as a convenient model for studying MSCs capable of transferring hematopoietic microenvironment under the renal capsule of syngeneic mice. BM from one femur of NES-EGFP mouse was used to obtain Dexter-type long-time bone marrow culture (LTBMCs, n = 5). GFP+ cells had been counted weekly over the entire area of the T25 flasks for 5 weeks after culture initiation using a fluorescence microscope. Four weeks after the initiation of LTBMCs, two day long time-lapse vital photography was performed. Ectopic foci of hematopoiesis under the renal capsule (n = 6) were obtained from the BM of NES-EGFP donors in syngeneic recipients. The number of C-kit+/–GFP+ cells in the formed foci was determined using FACS analysis. GFP+ cells with stromal phenotype in the adherent cell layer of LTBMCs had been detected during 5 weeks of cultivation. The stromal layer of LTBMC contained fibroblast-like GFP-low and GFP-hi cell subpopulations. GFP-low cells were scattered across the surface and predominated. GFP-hi cells were rare and formed colonies. Ectopic foci of hematopoiesis contained 2036 ± 734 C-kit–GFP+ stromal cells. Most of these cells had GFP-low phenotype. The cellularity of the focus strongly and positively correlated with the number of C-kit–GFP+ in it. The results show that C-kit–GFP+ BM cells of NES-EGFP mice are able to survive implantation and include MSCs and early stromal mesenchymal progenitors, the number of which determines the size of the focus. NES-EGFP mice can be used for in vitro and in vivo experiments to study functional BM-derived MSCs.

Biomimetic titanium implant coated with extracellular matrix enhances and accelerates osteogenesis.

Aim: To evaluate the biological function of titanium implants coated with cell-derived mineralized extracellular matrix, which mimics a bony microenvironment. Materials & methods: A biomimetic titanium implant was fabricated primarily by modifying the titanium surface with TiO2 nanotubes or sand-blasted, acid-etched topography, then was coated with mineralized extracellular matrix constructed by culturing bone marrow mesenchymal stromal cells. The osteogenic ability of biomimetic titanium surface in vitro and in vivo were evaluated. Results:In vitro and in vivo studies revealed that the biomimetic titanium implant enhanced and accelerated osteogenesis of bone marrow stromal cells by increasing cell proliferation and calcium deposition. Conclusion: By combining surface topography modification with biological coating, the results provided a valuable method to produce biomimetic titanium implants with excellent osteogenic ability.

Myocardial Patch Formation by Three-Dimensional Culture of Bone Marrow Mesenchymal Stem Cells with 3-Hydroxybutyrate-Co-4-Hydroxybutyrate Under Hypoxia

Herein we researched the effects of a hypoxic microenvironment on bone marrow mesenchymal stem cells (BM-MSCs) on poly 3-hydroxybutyrate-co-4-hydroxybutyrate [P(3HB-co-4HB)] and present a theoretical basis for development of cell transplantation. Mouse bone marrow mesenchymal stem cells were isolated by whole bone marrow culture and surface antigens were analyzed by flow cytometry of passage 5 cells. P(3HB-co-4HB) and bone marrow mesenchymal stem cells were prepared as stem cell patches randomly divided into normoxia (control, 20% oxygen) and hypoxia (3% oxygen) groups. After 24 h, the patch was used for experiments. Cell proliferation was determined by CCK-8 assays. Adhesion, survival, and growth of cells on patches were observed by scanning electron microscopy. Expression of hypoxia-inducible factor-1α (HIF-1α) was tested by real-time quantitative PCR and western blotting. At 2 weeks after addition of cardiomyocyte differentiation inducer 5-azacytidine, cardiac troponin T (cTnT) expression was detected by immunofluorescence. After 24 h, the proliferation of the hypoxic group was considerably greater compared with the normoxic group (n = 12,P < 0 05). SEM demonstrated that the number of viable cells in the hypoxic group was higher than that in the normoxic group. Adhesion between cells and the patch was firm and cell morphology was normal in the hypoxic group. Significant upregulation of HIF-1α mRNA was observed by real-time quantitative PCR after 12 h (P < 0 05). HIF-1αprotein expression in the hypoxia group was considerably higher than that in the normoxia group. cTnT expression in the hypoxic group was more pronounced than that in the normoxic group. Our results show that a hypoxic microenvironment promotes the adhesion, survival, proliferation, and myocardial differentiation of bone marrow mesenchymal stem cells on a P(3HB-co-4HB) patch, which may be mediated by the HIF-1α; pathway.

Bone Marrow-Derived Dendritic Cell Cultures from RAG-/- Mice Include IFN-γ-Producing NK Cells.

Dendritic cells (DCs) play a key role in the initiation of an immune response and are known as "professional" APCs because of their ability to activate naive T cells. A widely used method to generate DCs in vitro is to culture bone marrow (BM) cells or blood monocytes in the presence of GM-CSF and IL-4. In this study, we show that a small population of NK cells residing in the BM of RAG-/-, but not RAG-/- γc chain-/- mice, remain in the DC culture and is the source of IFN-γ produced after stimulation with LPS. These cells, which may represent early promoters of LPS-induced responses, have to be taken into account when interpreting experiments using BM-derived DCs.

Acoustophoresis Enables the Label-Free Separation of Functionally Different Subsets of Cultured Bone Marrow Stromal Cells.

Culture-expanded mesenchymal stromal cells (MSCs) are promising candidates for clinical cell-based therapies. MSC products are heterogeneous and we therefore investigated whether acoustophoresis, an ultrasound-based separation technology, could be used for the label-free enrichment of functionally different MSC populations. Acoustophoresis uses an ultrasonic standing wave field in a microchannel that differentially affects the movement of cells depending on their acoustophysical properties, such as size, density, and compressibility. Human bone marrow (BM) MSCs were generated by standard adherent culture in xeno-free medium and separated by microchip acoustophoresis. MSCs with up to 20% higher proliferation and 1.7-fold increased clonogenic potential were enriched in the side outlet of the chip compared to the input sample. These cells were significantly smaller (average diameter 14.5 ± 0.4μm) compared to the center outlet fraction (average diameter 17.1 ± 0.6μm) and expressed higher levels of genes related to proliferation and stem cell properties (i.e., Ki-67 [1.9-fold], Nanog1 [6.65-fold], Oct4 [2.9-fold], and CXCL12 [1.8-fold], n=3) in the side outlet compared to input. Fractions of MSCs in G0 /G1 cell cycle phase were significantly enriched in the side fraction and an up to 2.8-fold increase of cells in S/G2 /M phases were observed in center fractions compared to side fractions and 1.3-fold increased compared to the input sample. Acoustophoresis did not compromise MSC phenotype, proliferation, clonogenic capacity, and viability (generally 87-98%), nor did it affect differentiation or immunomodulatory capacities. These results demonstrate that label-free acoustic separation can enrich functionally different MSC subsets which can potentially be employed to produce better-defined stromal cell products from cultured MSCs. Hence, acoustophoresis is a potentially promising separation technology to provide improved cell products for research and possible future clinical use. © 2020 The Authors. Cytometry Part A published by Wiley Periodicals LLC. on behalf of International Society for Advancement of Cytometry.

Interplay Between the IL-33/ST2 Axis and Bone Marrow ILC2s in Protease Allergen-Induced IL-5-Dependent Eosinophilia.

Background: Eosinophils develop from CD34+ progenitor cells in the bone marrow under the influence of interleukin (IL)-5. Several cell types produce IL-5, including type 2 innate lymphoid cells (ILC2s). The alarmin cytokine IL-33 is known to activate ILC2s in mucosal tissues, but little is known about IL-33-responsive ILC2s in the bone marrow in allergen-induced airway inflammation.Methods: Wild type (WT) and Rag1 deficient (Rag1−/−) mice, which lack mature T and B cells, received intranasal doses of papain to induce acute allergic inflammation. In some experiments, mice were pre-treated with anti-IL-5 prior to the papain challenge. Furthermore, recombinant IL-33 was administered to WT mice, Rag1−/− mice, lymphocyte deficient mice (Rag2−/−Il2rg−/−) and to ex vivo whole bone marrow cultures. Bone marrow eosinophils and ILC2s were analyzed by flow cytometry. Eosinophil count was assessed by differential cell count and secreted IL-5 from bone marrow cells by ELISA.Results: Intranasal administration of papain or IL-33 increased the number of mature eosinophils in the bone marrow despite the absence of adaptive immune cells in Rag1−/− mice. In parallel, an increased number of eosinophils was observed in the airways together with elevated levels of Eotaxin-2/CCL24. Bone marrow ILC2s were increased after papain or IL-33 administration, whereas ILC2s was found to be increased at baseline in Rag1−/− mice compared to WT mice. An upregulation of the IL-33 receptor (ST2) expression on bone marrow ILC2s was observed after papain challenge in both Rag1−/− and WT mice which correlated to increased number of bone marrow eosinophilia. Furthermore, an increased number of ST2+ mature eosinophils in the bone marrow was observed after papain challenge, which was further dependent on IL-5. In addition, bone marrow-derived ILC2s from both mouse strains produced large amounts of IL-5 ex vivo after IL-33 stimulation of whole bone marrow cultures. In contrast, IL-33-induced bone marrow and airway eosinophilia were abolished in the absence of ILC2s in Rag2−/−Il2rg−/− mice and no production of IL-5 was detected in IL-33-stimulated bone marrow cultures.Conclusion: These findings establish bone marrow ILC2s and the IL-33/ST2 axis as promising targets for modulation of uncontrolled IL-5-dependent eosinophilic diseases including asthma.

Isolation and characterization of endothelial progenitor cells from canine bone marrow

ABSTRACT Endothelial progenitor cells (EPC) are located predominantly in the bone marrow. These cells are useful for treating human vascular diseases; they also are a possible target for restricting blood vessel growth for tumors. Little is known about canine EPC. We investigated a bone marrow EPC isolation method that combines the whole bone marrow culture method and the differential adherent speed method using stillborn canines. MTT proliferation, flow cytometry detection, Dil-ac-LDL uptake, FITC-UEA-1 binding and matrigel assays were used to identify and characterize EPC. We isolated two types of EPC: early EPC and late EPC. We found that isolated cells produced typical colony and cobblestone morphology, and were positive for CD31, CD34, CD133 and VEGFR-2. Significant differences were observed in the intensity of expression between early and late EPC, which suggests their different roles during angiogenesis and vasculogenesis. Both early and late EPC were positive for Dil-ac-LDL and FITC-UEA-1, and displayed tube formation when re-suspended in matrigel, both of which are important functional criteria for identifying EPC. Our method is a novel, effective and efficient way to produce enriched EPC.

P0530A NOVEL LNCRNA(LRAN9884) PROMOTES AKI VIA MAINTAINING MINCLE-DEPENDENT M1 MACROPHAGE PHENOTYPE

Abstract Background and Aims Macrophages are key inflammatory cells and play a critical role in renal inflammation in acute kidney injury (AKI). M1 inflammatory macrophage and M2 anti-inflammatory macrophage act reverse role. Phenotype of macrophages is highly flexible and can be changed over time. However, the underlying mechanism of M1 and M2 macrophage phenotype switching during AKI is still largely unclear. In our previous study, we identified a novel lncRNA (LRNA9884) might contribute to inflammation according to modifying macrophages. The present study was designed to uncover the pathogenic role and the underlying mechanism of LRNA9884 in cisplatin-induced AKI. Method Expression level and pattern of LRNA9884 were examined in cisplatin-induced AKI mice. The regulatory mechanisms of LRNA9884 was investigated in cultured bone marrow–derived macrophages (BMDMs) in vitro by silencing or overexpressing of LRNA9884. Flow Cytometer, fluorescence in situ hybridization (FISH) and other multiple molecular biological techniques were applied to figure out the role of LRNA9884 under acute kidney injury. Results LRNA9884 was significantly upregulated in the kidney of cisplatin-induced mice and was associated with the progression of the renal inflammation by using RT-PCR and ISH assay. FISH assay with immunofluorescence co-staining detected that LRNA9884 was largely expressed in the nucleus of macrophage in cisplatin-induced mice kidney compared with the sham group at day 1 after AKI injury. LRNA9884 was remarkedly induced by TNF-α (10ng/ml) in BMDMs as time- and dose- dependent. Western blot and RT-PCR showed that silencing of LRNA9884 effectively inhibited upregulated of macrophage-inducible C-type lectin (Mincle) and iNOS induced by TNF-α. More importantly, we identified that LRNA9884 maintained M1 macrophages phenotype by triggering mincle production at transcriptional level as evidenced by ChIP assay. Conclusion LRNA9884 is a mincle-dependent lncRNA that highly-expressed in macrophages under AKI development. Targeting of LRNA9884 effectively blocked the inflammatory response via promoting the transition M1 macrophage to M2 macrophage phenotype. This study will shed new lights on the understanding of pathological role of novel LRNA9884 in AKI.

The effect of conditioned media on mouse oocytes ultrastructure following in vitro maturation

Optimizing the culture condition and vitrification process during in vitro maturation (IVM) of oocyte has been controversial. This study aimed to evaluate the effect conditioned medium (CM) enriched with human mesenchymal stem cells (hUCM) derived from bone marrow and umbilical cord blood on the ultrastructure of immature mouse oocytes during IVM. Three hundred ninety-two germinal vesicle (GV) oocytes were obtained from mouse ovaries. They were divided into fresh IVM (fIVM) and vitrified IVM (vIVM) groups and cultivated in either alpha Minimum Essential Medium (α-MEM) (control) or CMs supplemented with supernatants derived from cultures of bone marrow or umbilical cord mesenchyme stem cells (MSCs). Then the maturation rate was evaluated in the experimental groups. Twelve in vivo fresh mature oocytes were randomly obtained from uterine tubes as the control group. Finally, 43 matured IVM oocytes were randomly selected for ultra-structural evaluations using transmission electron microscopy (TEM). Oocyte maturation rates were significantly higher in fIVM than vIVM groups. The highest maturation rate was observed in the fIVM umbilical cord MSCs while the lowest rate was related to α-MEM vIVM (86.44% vs 72.72%). There was an increase in the number of mitochondria–vesicle (MV) complexes in IVM groups. Also, the distribution of cortical granules showed the highest decline in α-MEM vIVM groups. The highest aggregations of mitochondria–SER (M-SER) were observed in fIVM umbilical cords. Oolemma had an irregular border with small microvilli in vIVM groups. The Zona pellucida was darker in the vIVM than fIVM groups. In conclusion, umbilical cord MSCs showed a superior efficacy than bone marrow cells for supporting oocyte maturation and conserving its ultrastructure.

The Amino Acid Sensor Eif2ak4/GCN2 Is Required for Proliferation of Osteoblast Progenitors in Mice.

Skeletal stem/progenitor cells (SSPC) are critical regulators of bone homeostasis by providing a continuous supply of osteoblasts throughout life. In response to inductive signals, SSPC proliferate before osteoblast differentiation. Proliferation requires the duplication of all cellular components before cell division. This imposes a unique biosynthetic requirement for amino acids that can be used for biomass production. Thus, the ability to sense and respond to amino acid availability is likely a major determinant for proliferation. Using a cellular and genetic approach, we demonstrate the amino acid sensor GCN2 is required to support the robust proliferative capacity of SSPC during bone homeostasis. GCN2 ablation results in decreased postnatal bone mass due primarily to reduced osteoblast numbers. Decreased osteoblast numbers is likely attributed to reduced SSPC proliferation as loss of GCN2 specifically affected proliferation in cultured bone marrow stromal cells (BMSCs) without impacting osteoblast differentiation in vitro. Mechanistically, GCN2 regulates proliferation by increasing amino acid uptake downstream of the transcriptional effector ATF4. Collectively, these data suggest amino acid sensing through the GCN2/ATF4 pathway is indispensable for robust SSPC proliferation necessary for bone homeostasis. © 2020 American Society for Bone and Mineral Research.

Systemic but not MDSC-specific IRF4 deficiency promotes an immunosuppressed tumor microenvironment in a murine pancreatic cancer model

Pancreatic ductal adenocarcinoma is characterized by a strong immunosuppressive network with a dense infiltration of myeloid cells including myeloid-derived suppressor cells (MDSC). Two distinct populations of MDSC have been defined: polymorphonuclear MDSC (PMN-MDSC) and monocytic MDSC (M-MDSC). Several factors influence the development and function of MDSC including the transcription factor interferon regulatory factor 4 (IRF4). Here, we show that IRF4 deficiency accelerates tumor growth and reduces survival, accompanied with a dense tumor infiltration with PMN-MDSC and reduced numbers of CD8+ T cells. As IRF4 has been described to modulate myeloid cell development and function, particularly of PMN-MDSC, we analyzed its role using MDSC-specific IRF4 knockout mice with the Ly6G or LysM knock-in allele expressing Cre recombinase and Irf4flox. In GM-CSF-driven bone marrow cultures, IRF4 deficiency increased the frequency of MDSC-like cells with a strong T cell suppressive capacity. Myeloid (LysM)-specific depletion of IRF4 led to increased tumor weight and a moderate splenic M-MDSC expansion in tumor-bearing mice. PMN cell (Ly6G)-specific depletion of IRF4, however, did not influence tumor progression or MDSC accumulation in vivo in accordance with our finding that IRF4 is not expressed in PMN-MDSC. This study demonstrates a critical role of IRF4 in the generation of an immunosuppressive tumor microenvironment in pancreatic cancer, which is independent of IRF4 expression in PMN-MDSC.

Silibinin Upregulates CXCR4 Expression in Cultured Bone Marrow Cells (BMCs) Especially in Pulmonary Arterial Hypertension Rat Model.

Previously we reported that silibinin ameliorated pulmonary arterial hypertension (PAH) in rat PAH models, possibly through the suppression of the CXCR4/SDF-1, until the point where PAH became a severe and irreversible condition. To further investigate how silibinin ameliorates PAH, we first attempted to clarify its effect on bone marrow cells (BMCs), since the CXCR4/SDF-1 axis is known to regulate stem cell migration and attachment in BM niches. Rat PAH models were established through a combination of a single subcutaneous injection of monocrotaline (MCT) and chronic hypoxic conditions (10% O2). BMCs were harvested and cultured, and reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and flow cytometry (FCM) were performed to investigate whether silibinin affected CXCR4 expression. Silibinin upregulated the gene expression of stem cell related markers CXCR4, SDF-1, SCF, and c-Kit, inflammatory markers IL-6 and TNFα, mesenchymal stem cell (MSC)-related markers CD44 and CD29, and the granulocyte/monocyte-macrophage marker CD14 in cultured BM in PAH rats, but not in normal rats, except CXCR4. FCM showed that silibinin increased the CXCR4-positive cell population in a granulocyte fraction of cultured BMCs. However, immunohistochemical (IHC) staining showed no significant change in CXCR4 expression in the BM of the tibias. These results suggest that silibinin increases the expression of CXCR4 in BM, and the increased CXCR4-positive cells could be granulocytes/monocyte-macrophages.

Identification of dendritic cell precursors in the culture of bone marrow with hematopoietic cytokines

Abstract Dendritic cells (DCs) play crucial parts in a variety of immune responses by capturing and presenting antigens to naïve T cells and by producing the subsequent T-cell activation or tolerance. In vitro culture systems of DCs have been widely used. The culture of bone marrow (BM) in the presence of a hematopoietic cytokine, Fms-like tyrosine kinase 3 ligand (FLT3L), produces plasmacytoid DCs (pDCs) and classical DCs (cDCs), whereas the culture of BM with granulocyte-macrophage colony-stimulating factor (GM-CSF) generates DCs and macrophages considered to possess inflammatory phenotypes. In the present study, we examined the culture of BM with both cytokines, i.e., FLT3L and GM-CSF in combination and identified 4 different subsets in CD11c⁺ MHCII+ cells. Meanwhile, in the culture of BM with FLT3L, we discovered a novel population of CD11c⁺ MHCIIhi cells that possess a limited capacity of antigen-presenting activity, suggesting precursors for DCs. This novel population of DC precursors from the BM culture with FLT3L was isolated and evaluated for their potential to differentiate to DCs in the BM culture containing both FLT3L and GM-CSF. Consequently, we demonstrated that the presence of a novel population of DC precursors in the in vitro culture system of DCs.

Bone microenvironment-suppressed T cells increase osteoclast formation and breast cancer bone metastases

Abstract Bone metastases is a frequent (>75%) complication of advanced breast cancer (BCa). BCa cells in bone are supported by cytokines released during the osteoclastic resorption, and remain untreatable. T cells during immunotherapy could turn against BCa cells in bone but could also increase osteoclasts and bone metastases. Comparing the development of bone metastases from 4T1 BCa cells between Balb/C and SCID or T cell-depleted mice, the absence of T cells decreased bone metastases (−72% and −38%, respectively), while orthotopic tumors were increased. Histology confirmed that the osteoclast number was decreased at the tumor-bone interface of mice lacking T cells. Ex vivo addition of non-activated T cells increased osteoclast formation, and flow cytometry confirmed that >85% of T cells in bone metastases were not expressing activation markers. In sharp contrast, activated T cells were potent inhibitors of osteoclast formation, which could be beneficial for the treatment of patients. Although activated T cells expressed the anti-osteoclastic Ifng and Il4, their effect was not reversed by neutralizing antibodies, or due to a bystander effect by cytotoxic T cells. Thus, we sought to activate T cells of bone metastases. However, it was not possible to activate T cells in bone marrow cultures ex vivo. This could be due to an increase of MDSCs in 4T1 bone metastases, including M-MDSCs that were PD-L1+ (>85%) and could suppress PD-1+ T cells (>70%) in bone. Our results suggest that while T cells under the influence of the bone microenvironment promote BCa bone metastases, immunotherapy-activated T cells could suppress bone resorption and eliminate BCa cells in patients with bone metastases.