Altered expression of circadian clock genes during peripheral blood stem cell mobilization induced by granulocyte colony-stimulating factor

Circulating hematopoietic stem cells exhibit robust circadian fluctuations, which influence the mobilized cell yield, even during enforced stem cell mobilization. However, alterations in the expression of circadian clock genes during granulocyte colony-stimulating factor (G-CSF)-induced peripheral blood stem cell (PBSC) mobilization are not fully elucidated. Therefore, we measured the expression of these genes in human peripheral blood leukocytes from 21 healthy donors. While CRY1 mRNA expression significantly increased by 3.9-fold (p?<?0.01), the expression of PER3, CRY2 and BMAL1 mRNAs significantly decreased (by 0.2-fold, 0.2-fold, and 0.6-fold, respectively; p?<?0.001) after G-CSF administration. Moreover, CRY1 mRNA expression was inversely correlated with the plasma level of noradrenaline (r?=??0.36, p?<?0.05), while PER3, CRY2, and BMAL1 mRNA expression directly correlated with the plasma level of noradrenaline (r?=?0.55, r?=?0.66, and r?=?0.57, respectively; p?<?0.001). Thus, significant correlations between the levels of circadian clock gene mRNAs and the plasma level of noradrenaline, a sympathetic nervous system neurotransmitter, were established. The modulation of sympathetic activation and of the circadian clock may be novel therapeutic targets for increasing stem cell yields in PBSC donors.     

The hypoxic peri-arteriolar glioma stem cell niche,an integrated concept of five types of niches in human glioblastoma

Glioblastoma is the most lethal primary brain tumor and poor survival of glioblastoma patients is attributed to the presence of glioma stem cells (GSCs). These therapy-resistant, quiescent and pluripotent cells reside in GSC niches, which are specific microenvironments that protect GSCs against radiotherapy and chemotherapy. We previously showed the existence of hypoxic peri-arteriolar GSC niches in glioblastoma tumor samples. However, other studies have described peri-vascular niches, peri-hypoxic niches, peri-immune niches and extracellular matrix niches of GSCs. The aim of this review was to critically evaluate the literature on these five different types of GSC niches. In the present review, we describe that the five niche types are not distinct from one another, but should be considered to be parts of one integral GSC niche model, the hypoxic peri-arteriolar GSC niche. Moreover, hypoxic peri-arteriolar GSC niches are structural and functional look-alikes of hematopoietic stem cell (HSC) niches in the bone marrow. GSCs are maintained in peri-arteriolar niches by the same receptor-ligand interactions as HSCs in bone marrow. Our concept should be rigidly tested in the near future and applied to develop therapies to expel and keep GSCs out of their protective niches to render them more vulnerable to standard therapies.     

Expansion of NK cells by engineered K562 cells co-expressing 4-1BBL and mMICA,combined with soluble IL-21

NK cells hold promise for protecting hosts from cancer and pathogen infection through direct killing and expressing immune-regulatory cytokines. In our study, a genetically modified K562 cell line with surface expression of 4-1BBL and MICA was constructed to expand functional NK cells in vitro for further adoptive immunotherapy against cancer. After a long-term up to 21 day co-culture with newly isolated peripheral blood mononuclear cells (PBMCs) in the presence of soluble IL-21 (sIL-21), notable increase in proportion of expanded NK cells was observed, especially the CD56^brightCD16⁺ subset. Apparent up-regulation of activating receptors CD38, CD69 and NKG2D was detected on expanded NK cells, so did inhibitory receptor CD94; the cytotoxicity of expanded NK cells against target tumor cells exceeded that of NK cells within fresh PBMCs. The intracellular staining showed expanded NK cells produced immune-regulatory IFN-γ. Taken together, we expanded NK cells with significant up-regulation of activating NKG2D and moderate enhancement of cytotoxicity, with IFN-γ producing ability and a more heterogeneous population of NK cells. These findings provide a novel perspective on expanding NK cells in vitro for further biology study and adoptive immunotherapy of NK cells against cancer.     

Elevated granulocyte-colony stimulating factor and hematopoietic stem cell mobilization in Niemann-Pick type C1 disease

Niemann-Pick type C1 (NPC1) disease is a progressive lysosomal storage disorder caused by mutations of the NPC1 gene. While neurodegeneration is the most severe symptom, a large proportion of NPC1 patients also present with splenomegaly, which has been attributed to cholesterol and glycosphingolipid accumulation in late endosomes and lysosomes. However, recent data also reveal an increase in the inflammatory monocyte subset in the Npc1^nih mouse model expressing an Npc1 null allele. We evaluated the contribution of hematopoietic cells to splenomegaly in NPC1 disease under conditions of hypercholesterolemia. We transplanted Npc1^nih (Npc1 null mutation) or Npc1^wt bone marrow (BM) into Ldlr^?/? mice and fed these mice a cholesterol-rich Western-type diet. At 9 weeks after BM transplant, on a chow diet, the Npc1 null mutation increased plasma granulocyte-colony stimulating factor (G-CSF) by 2-fold and caused mild neutrophilia. At 18 weeks after BM transplant, including 9 weeks of Western-type diet feeding, the Npc1 mutation increased G-csf mRNA levels by ～5-fold in splenic monocytes/macrophages accompanied by a ～4-fold increase in splenic neutrophils compared with controls. We also observed ～5-fold increased long-term and short-term hematopoietic stem cells (HSCs) in the spleen, and a ～30–75% decrease of these populations in BM, reflecting HSC mobilization, presumably downstream of elevated G-CSF. In line with these data, four patients with NPC1 disease showed higher plasma G-CSF compared with age-matched and gender-matched healthy controls. In conclusion, we show elevated G-CSF levels and HSC mobilization in the setting of an Npc1 null mutation and propose that this contributes to splenomegaly in patients with NPC1 disease.     

Role of cytokines and extracellular matrix in the regulation of haemopoietic stem cells

The understanding of molecular mechanisms regulating the formation, growth and differentiation of haemopoietic stem cells has advanced considerably recently. Particular progress has been made in defining the cytokines, chemokines and extracellular matrix components which retain and maintain primitive haemopoietic cell populations in bone marrow. Furthermore, signal transduction pathways that are critical for haemopoiesis, both in vivo and in vitro, and that are activated by cytokines have also been identified and further characterised. The importance of these processes has, this year, been exemplified by the phenotypes of mice deficient in key signal transduction proteins and the discovery that mutations in the component proteins of some signalling pathways are linked to human diseases. Significant advances in understanding the molecular mechanisms for mobilisation of stem cells from bone marrow have also been made this year; this has potential importance for bone marrow transplantation.     

Reformation in chimeric antigen receptor based cancer immunotherapy: Redirecting natural killer cell

Natural killer (NK) cells are an important subset of lymphocytes which play a critical role in host immunity against cancers. With MHC-independent recognition, short lifespan and potent cytotoxicity, NK cells make a promising candidate for chimeric antigen receptor (CAR)-engineered cancer immunotherapy. Due to innate biological properties of NK cells, CAR-NK may outperform CAR-T therapy in terms of less side effects and more universal access, which may become a great reformation in CAR-based cancer immunotherapy. The CARs used in peripheral blood (PB) NK cells as well as NK cell line like NK-92 are the most important outfits defining antigenic specificity. The constructs of CARs used in NK cells from different sources vary, which all undergo generational optimization. The anti-tumor effects of CAR-NK have been validated in numerous preclinical trials for cancers, including hematologic malignancies and many solid tumors, which provide evidence for potential clinical application of CAR-NK. Additionally, this review concludes the challenges faced in the application of CAR-NK. Although CAR-NK is considered as one of the most possible “off-the-shelf” products, the improvement for the efficiency of expansion and transduction as well as the solution for underlying safety issues is still needed. Possible coping strategies for challenges and upgrades in techniques are also highlighted for future development in CAR-NK cancer immunotherapy.     

Tumor necrosis factor alpha promotes the expression of immunosuppressive proteins and enhances the cell growth in a human bone marrow-derived stem cell culture

Mesenchymal stem cells (MSCs) are widely used in experimental treatments for various conditions that involve normal tissue regeneration via inflammatory repair. It is known that MSCs can secrete multiple soluble factors and suppress inflammation. Even though the effect of MSCs on inflammation has been extensively studied, the effect of inflammation on MSCs is poorly understood. One of the major cytokines released at the site of inflammation is tumor necrosis factor alpha (TNF-α) which is known to induce MSC invasion and proliferation. Therefore, we wanted to test the effects of TNF-α exposure on MSCs derived from human bone marrow. We found, as expected, that cell proliferation was significantly enhanced during TNF-α exposure. However, according to the cell surface marker analysis, the intensity of several antigens in the minimum criteria panel for MSCs proposed by International Society of Cellular Therapy (ISCT) was decreased dramatically, and in certain cases, the criteria for MSCs were not fulfilled. In addition, TNF-α exposure resulted in a significant but transient increase in human leukocyte antigen and CD54 expression. Additional proteomic analysis by two-dimensional difference gel electrophoresis and mass spectrometry revealed three proteins whose expression levels decreased and 8 proteins whose expression levels increased significantly during TNF-α exposure. The majority of these proteins could be linked to immunosuppressive and signalling pathways. These results strongly support reactive and immunosuppressive activation of MSCs during TNF-α exposure, which might influence MSC differentiation stage and capacity.     

Review: In vitro generation of red blood cells for transfusion medicine: Progress,prospects and challenges

In vitro generation of red blood cells (RBCs) has the potential to circumvent the shortfalls in global demand for blood for transfusion applications. The conventional approach for RBC generation has been from differentiation of hematopoietic stem cells (HSCs) derived from cord blood, adult bone marrow or peripheral blood. More recently, RBCs have been generated from human induced pluripotent stem cells (hiPSCs) as well as from immortalized adult erythroid progenitors. In this review, we highlight the recent advances to RBC generation from these different approaches and discuss the challenges and new strategies that can potentially make large-scale in vitro generation of RBCs a feasible approach.     

Cannabinoid receptor signaling in progenitor/stem cell proliferation and differentiation

Cannabinoids, the active components of cannabis (Cannabis sativa) extracts, have attracted the attention of human civilizations for centuries, much earlier than the discovery and characterization of their substrate of action, the endocannabinoid system (ECS). The latter is an ensemble of endogenous lipids, their receptors [in particular type-1 (CB₁) and type-2 (CB₂) cannabinoid receptors] and metabolic enzymes. Cannabinoid signaling regulates cell proliferation, differentiation and survival, with different outcomes depending on the molecular targets and cellular context involved. Cannabinoid receptors are expressed and functional from the very early developmental stages, when they regulate embryonic and trophoblast stem cell survival and differentiation, and thus may affect the formation of manifold adult specialized tissues derived from the three different germ layers (ectoderm, mesoderm and endoderm). In the ectoderm-derived nervous system, both CB₁ and CB₂ receptors are present in neural progenitor/stem cells and control their self-renewal, proliferation and differentiation. CB₁ and CB₂ show opposite patterns of expression, the former increasing and the latter decreasing along neuronal differentiation. Recently, endocannabinoid (eCB) signaling has also been shown to regulate proliferation and differentiation of mesoderm-derived hematopoietic and mesenchymal stem cells, with a key role in determining the formation of several cell types in peripheral tissues, including blood cells, adipocytes, osteoblasts/osteoclasts and epithelial cells. Here, we will review these new findings, which unveil the involvement of eCB signaling in the regulation of progenitor/stem cell fate in the nervous system and in the periphery. The developmental regulation of cannabinoid receptor expression and cellular/subcellular localization, together with their role in progenitor/stem cell biology, may have important implications in human health and disease.     

MicroRNAs and extracellular vesicles in cholangiopathies

Cholangiopathies encompass a heterogeneous group of disorders affecting biliary epithelial cells (i.e. cholangiocytes). Early diagnosis, prognosis and treatment still remain clinically challenging for most of these diseases and are critical for adequate patient care. In the past decade, extensive research has emphasized microRNAs (miRs) as potential non-invasive biomarkers and tools to accurately identify, predict and treat cholangiopathies. MiRs can be released extracellularly conjugated with lipoproteins or encapsulated in extracellular vesicles (EVs). Research on EVs is also gaining attention since they are present in multiple biological fluids and may represent a relevant source of novel non-invasive biomarkers and be vehicles for new therapeutic approaches. This review highlights the most promising candidate miRs and EV-related biomarkers in cholangiopathies, as well as their relevant roles in biliary pathophysiology. This article is part of a Special Issue entitled: Cholangiocytes in Health and Disease edited by Jesus Banales, Marco Marzioni, Nicholas LaRusso and Peter Jansen.