Marrow Components Research Articles

Antineoplastic therapy affects the in vitro phenotype and functionality of healthy human bone marrow-derived mesenchymal stromal cells.

While antineoplastic therapies aim to specifically target cancer cells, they may also exert adverse effects on healthy tissues, like healthy hematopoietic stem and progenitor cells (HSPC), leading to hematotoxicity as a common side effect. Mesenchymal stromal cells (MSC) are a major component of the bone marrow (BM) microenvironment, regulating normal hematopoiesis, while their susceptibility to anticancer therapies and contribution to therapy-related hematotoxicity remains largely unexplored. To address this, we investigated the effects of etoposide, temozolomide, 5-azacitidine, and venetoclax on healthy BM-derived MSC functionality. Doses below therapeutic effects of etoposide (0.1-0.25µM) inhibited cellular growth and induced cellular senescence in healthy MSC, accompanied by an increased mRNA expression of CDKN1A, decreased trilineage differentiation capacity, and insufficient hematopoietic support. Pharmacological doses of 5-azacitidine (2.5µM) shifted MSC differentiation capacity by inhibiting osteogenic capacity but enhancing the chondrogenic lineage, as demonstrated by histochemical staining and on mRNA level. At the highest clinically relevant dose, neither venetoclax (40nM) nor temozolomide (100µM) exerted any effects on MSC but clearly inhibited cellular growth of cancer cell lines and primary healthy HSPC, pointing to damage to hematopoietic cells as a major driver of hematotoxicity of these two compounds. Our findings show that besides HSPC, also MSC are sensitive to certain antineoplastic agents, resulting in molecular and functional alterations that may contribute to therapy-related myelosuppression. Understanding these interactions could be helpful for the development of strategies to preserve BM MSC functionality during different kinds of anticancer therapies.

Abstracts BMAS Summer School 2023-2nd Bone Marrow Adiposity Society Summer School Meeting 2023.

Fat is the main component of an adult bone marrow and constitutes the so-called bone marrow adipose tissue (BMAT). Marrow adipocytes, which are the fat cells in the bone marrow, become more abundant with age, and may influence the whole-body metabolism. In osteoporotic patients, the amount of BMAT has an inverse correlation with the amount of bone mass. In people with anorexia nervosa that lose weight after the reduction of peripheral adipose tissues, BMAT expands. Although bone marrow adipocytes are increasingly recognized as a target for therapy, there is still much to learn about their role in skeletal homeostasis, metabolism, cancer, and regenerative treatments. The Bone Marrow Adiposity Society (BMAS), established in 2017, aims to enhance the understanding of how BMAT relates to bone health, cancer, and systemic metabolism. BMAS is committed to training young scientists and organized the second edition of the BMAS Summer School, held on September 4-6, 2023, as a virtual event.

Machine learning-mediated identification of ferroptosis-related genes in osteonecrosis of the femoral head.

Osteonecrosis of the femoral head (ONFH) is a condition caused by a disruption or damage to the femoral head's blood supply, which causes the death of bone cells and bone marrow components and prevents future regeneration. Ferroptosis, a type of controlled cell death, is caused by iron-dependent lipid peroxidation. Here, we identified ferroptosis-related genes and infiltrating immune cells involved in ONFH and predicted the underlying molecular mechanisms. The GSE123568 dataset was subjected to differential expression analysis to identify genes related to ferroptosis. Subsequently, GO and KEGG pathway enrichment analyses, as well as protein-protein interaction (PPI) network analysis, were conducted. Hub genes involved in ferroptosis were identified using machine learning and other techniques. Additionally, immune infiltration analysis and lncRNA-miRNA-mRNA network prediction analysis were performed. Finally, we determined whether ferroptosis occurred by measuring iron content. The hub genes were validated by ROC curve analysis and qRT-PCR. Four ferroptosis-related hub genes (MAPK3, PTGS2, STK11, and SLC2A1) were identified. Additionally, immune infiltration analysis revealed a strong correlation among ONFH, hub genes, and various immune cells. Finally, we predicted the network relationship between differentially expressed lncRNAs and hub genes in the lncRNA-miRNA-mRNA network. MAPK3, PTGS2, STK11, and SLC2A1 have been identified as potential ferroptosis-related biomarkers and drug targets for the diagnosis and prognosis of ONFH, while some immune cells, as well as the interaction between lncRNA, miRNA, and mRNA, have also been identified as potential pathogenesis markers and therapeutic targets.

Biomaterials for bone defect repair: Types, mechanisms and effects.

Bone defects or bone discontinuities caused by trauma, infection, tumours and other diseases have led to an increasing demand for bone grafts and biomaterials. Autologous bone grafts, bone grafts with vascular tips, anastomosed vascular bone grafts and autologous bone marrow components are all commonly used in clinical practice, while oversized bone defects require the use of bone tissue engineering-related biomaterials to repair bone defects and promote bone regeneration. Currently, inorganic components such as polysaccharides and bioceramics, as well as a variety of bioactive proteins, metal ions and stem cells can be loaded into hydrogels or 3D printed scaffold materials to achieve better therapeutic results. In this review, we provide an overview of the types of materials, applications, potential mechanisms and current developments in the repair of bone defects.

Potentiality of Bone Marrow-Derived Mesenchymal Stromal Cells (BM-MSCs) to Differentiate into the Osteogenic Lineage in in Vitro Model of Tissue Regeneration

Introduction:The mesenchymal stromal cells (MSCs) have gained considerable popularity owing to the vast possibilities and lack of ethical constraints. MSCs serve as multipotent progenitors for several niche components in the bone marrow (BM) and play an essential role in the quiescence, proliferation, and differentiation of hematopoietic stem cells. Recently, MSCs from BM (BM-MSCs) represent an attractive cell source for tissue engineering, thanks to their immunomodulatory property, self-renewing and high proliferative capability. BM-MSCs have multi-lineages (adipogenesis, chondrogenesis, osteogenesis) potential and in combination with biomaterials, well support cell-attachments and stimulate extracellular matrix synthesis. For these reasons, BM-MSCs have a great potential for regenerative medicine applications. The present research aims to evaluate the ability of BM-MSCs to proliferate and differentiate toward the osteogenic lineage into innovative three-dimensional bioresorbable scaffold based on gelatin-chitosan hydrogel with a poly(lactic acid) lattice structure (PLA-CH) for regenerative medicine applications in the presence of fetal bovine serum (FBS) or human platelet lysate (HPL) with or without the osteogenic medium (OM). Methods: BM-MSCs were expanded to the passages 3 or 4 and seeded into the PLA-CH, in dry state, at a cellular density of 7x10 5 cells/scaffolds in growth medium (GM). For inducing osteogenic differentiation, cells/scaffolds constructs were cultured in 24-well plates for 4 weeks with OM consisting of a high-glucose DMEM supplemented with 10% FBS or 5% HPL, 10 −7M Dex, 25 mg/ml l-ascorbic acid, and 3mM NaH 2PO 4. For histomorphological analysis at optical microscope, scaffolds were embedded in paraffin using an automatic processor Donatello series 2 (Diapath S.p.A., Bergamo, Italy). Serial paraffin sections (5 µm thick) of each sample were cut, deparaffinized, and rehydrated, according to standard procedures and stained with hematoxylin-eosin stain for general morphology and Von Kossa (Bio-Optica, Milan, Italy) for calcium deposition. Immunohistochemistry for the osteogenic marker Osteocalcin (OSC) (Santa Cruz Biotechnologies, USA) was also performed. For Scanning Electron Microscopy (SEM) (EVO LS-10 manufactured by ZEISS) observation, samples have been progressively dehydrated through immersion in alcohol solutions. The local changes in elemental composition were investigated using the Energy Dispersive X-ray (EDX) analyzer. Results: The results showed that BM-MSCs have higher affinity to attachment, migrate and differentiate toward the osteogenic lineage inside the novel scaffold PLA-CH both with HPL and FBS as supplement in the culture media. The use of HPL for cell expansion offers the possibility to obtain a safer cellular products without xenogenic contaminants. Cells develop a large spreading area with elongated fibroblast-like morphology preferentially inside the hydrogel pores, showing great affinity with the scaffold. This is confirmed also by histomorphological analyses at optical microscope, showing homogeneous colonization of cells inside the hydrogel pores (figure 1a). The results showed the presence of calcium deposits in the mash of the scaffold with Von Kossa stain (figure 1b), moreover cells appeared well integrated with a positivity for OSC showing a clear differentiation state as osteoblasts (figure 1c). Calcium and phosphorous were detected with SEM-EDX on the samples incubated in osteogenic medium (figure 1 d,e). Conclusion: MSCs, derived from bone marrow, associated with scaffold PLA-CH proved to be biocompatible and promising for personalized medicine. In fact, BM-MSCs were able to growth, colonize and osteo-differentiate throughout the hydrogel, demonstrating their potential application for bone regenerative medicine. Figure 1. Histomorphological analysis at the optical microscope of PLA-CH with BM-hMSCs in the GM at 100 m m ( a), Von Kossa stain showing calcium deposit distribution on PLA-CH with BM-hMSCs in the OM at 30 m m ( b), immunohistochemistry for OCN with BM-hMSCs differentiated to osteoblast in the OM at 10 m m ( c). Evaluation of calcium (Ca) and phosphorous (P) with SEM-EDX of PLA-CH with BM-hMSCs in OM day 28 ( d,e).

Macrophage depletion using clodronate liposomes reveals latent dysfunction of the hematopoietic microenvironment associated with persistently imbalanced M1/M2 macrophage polarization in a mouse model of hemophagocytic lymphohistiocytosis.

Hemophagocytic lymphohistiocytosis (HLH), a hyperinflammatory syndrome, is caused by the incessant activation of lymphocytes and macrophages, resulting in damage to organs, including hematopoietic organs. Recently, we demonstrated that repeated lipopolysaccharide (LPS) treatment induces HLH-like features in senescence-accelerated (SAMP1/TA-1) mice but not in senescence-resistant control (SAMR1) mice. Hematopoietic failure in LPS-treated SAMP1/TA-1 mice was attributed to hematopoietic microenvironment dysfunction, concomitant with severely imbalanced M1 and M2 macrophage polarization. Macrophages are a major component of the bone marrow (BM) hematopoietic microenvironment. Clodronate liposomes are useful tools for in vivo macrophage depletion. In this study, we depleted macrophages using clodronate liposomes to determine their role in the hematopoietic microenvironment in SAMP1/TA-1 and SAMR1 mice. Under clodronate liposome treatment, the response between SAMR1 and SAMP1/TA-1 mice differed as follows: (1) increase in the number of activated M1 and M2 macrophages derived from newly generated macrophages and M2-dominant and imbalanced M1 and M2 macrophage polarization in the BM and spleen; (2) severe anemia and thrombocytopenia; (3) high mortality rate; (4) decrease in erythroid progenitors and B cell progenitors in the BM; and (5) decrease in the mRNA expression of erythroid-positive regulators such as erythropoietin and increase in that of erythroid- and B lymphoid-negative regulators such as interferon-γ in the BM. Depletion of residual macrophages in SAMP1/TA-1 mice impaired hematopoietic homeostasis, particularly erythropoiesis and B lymphopoiesis, owing to functional impairment of the hematopoietic microenvironment accompanied by persistently imbalanced M1/M2 polarization. Thus, macrophages play a vital role in regulating the hematopoietic microenvironment to maintain homeostasis.

Fresh Umbilical Cord Blood-A Source of Multipotent Stem Cells, Collection, Banking, Cryopreservation, and Ethical Concerns.

Umbilical cord blood (UCB) is a rich source of hematopoietic cells that can be used to replace bone marrow components. Many blood disorders and systemic illnesses are increasingly being treated with stem cells as regenerative medical therapy. Presently, collected blood has been stored in either public or private banks for allogenic or autologous transplantation. Using a specific keyword, we used the English language to search for relevant articles in SCOPUS and PubMed databases over time frame. According to our review, Asian countries are increasingly using UCB preservation for future use as regenerative medicine, and existing studies indicate that this trend will continue. This recent literature review explains the methodology of UCB collection, banking, and cryopreservation for future clinical use. Between 2010 and 2022, 10,054 UCB stem cell samples were effectively cryopreserved. Furthermore, we have discussed using Mesenchymal Stem Cells (MSCs) as transplant medicine, and its clinical applications. It is essential for healthcare personnel, particularly those working in labor rooms, to comprehend the protocols for collecting, transporting, and storing UCB. This review aims to provide a glimpse of the details about the UCB collection and banking processes, its benefits, and the use of UCB-derived stem cells in clinical practice, as well as the ethical concerns associated with UCB, all of which are important for healthcare professionals, particularly those working in maternity wards; namely, the obstetrician, neonatologist, and anyone involved in perinatal care. This article also highlights the practical and ethical concerns associated with private UCB banks, and the existence of public banks. UCB may continue to grow to assist healthcare teams worldwide in treating various metabolic, hematological, and immunodeficiency disorders.

Nutrient regulation of bone marrow adipose tissue: skeletal implications of weight loss.

Adipose tissue is a dynamic component of the bone marrow, regulating skeletal remodelling and secreting paracrine and endocrine factors that can affect haematopoiesis, as well as potentially nourishing the bone marrow during periods of stress. Bone marrow adipose tissue is regulated by multiple factors, but particularly nutrient status. In this Review, we examine how bone marrow adipocytes originate, their function in normal and pathological states and how bone marrow adipose tissue modulates whole-body homoeostasis through actions on bone cells, haematopoietic stem cells and extra-medullary adipocytes during nutritional challenges. We focus on both rodent models and human studies to help understand the unique marrow adipocyte, its response to the external nutrient environment and its effects on the skeleton. We finish by addressing some critical questions that to date remain unanswered.

Mdm2/p53 levels in bone marrow mesenchymal stromal cells are essential for maintaining the hematopoietic niche in response to DNA damage

Mesenchymal stromal cells (MSCs) are a key component of the bone marrow (BM) niche, providing essential support required for the maintenance of hematopoietic stem cells. To advance our understanding of physiological functions of p53 and Mdm2 in BM-MSCs, we developed traceable conditional mouse models targeting Mdm2 and/or Trp53 in vivo. We demonstrate that Mdm2 is essential for the emergence, maintenance, and hematopoietic support of BM-MSCs. Mdm2 haploinsufficiency in BM-MSCs resulted in genotoxic stress–associated thrombocytopenia, suggesting a functional role for Mdm2 in hematopoiesis. In a syngeneic mouse model of acute myeloid leukemia (AML), Trp53 deletion in BM-MSCs improved survival, and protected BM against hematopoietic toxicity from a murine Mdm2i, DS-5272. The transcriptional changes were associated with dysregulation of glycolysis, gluconeogenesis, and Hif-1α in BM-MSCs. Our results reveal a physiologic function of Mdm2 in BM-MSC, identify a previously unknown role of p53 pathway in BM-MSC–mediated support in AML and expand our understanding of the mechanism of hematopoietic toxicity of MDM2is.

Detection and Classification of Leukemia And Myeloma Using Soft Computing Techniques

Abstract: Blood cancer is a type of cancer that affects the blood cells. Medical image processing technology is essential in both early disease identification and cancer cell analysis. Blood cancer can impact the lymph nodes, bone marrow, blood cells, lymph nodes, and other lymphatic system components. A primary cause of blood cancer is an unusual and excessive amount of white blood cellular proliferation. Traditional cancer cell detection is time-consuming and inaccurate to a large extent, hence an automated approach based on soft computing techniques is presented to predict cancer cell presence and identify two types of blood cancer which are leukemia and myeloma. The dataset for Myeloma is acquired from TCIA (The Cancer Imaging Archive) repository and the Leukemia dataset comes from Kaggle-Blood Cell Images, both of which are available to the public. The datasets are already pre-processed. In our study, we have compared different hybrid models like DenseNet with XGBoost, InceptionResNet with SVM, etc from which the combination of VGG-19 for feature selection and SVM for classification gives the best performance. We have achieved Classification Accuracy of 96.4%(0.964), Precision(0.964), F1 Score(0.964) and Recall(0.964) for SVM.

Megakaryocyte Secreted Factors Regulate Bone Marrow Niche Cells During Skeletal Homeostasis, Aging, and Disease.

The bone marrow microenvironment contains a diverse array of cell types under extensive regulatory control and provides for a novel and complex mechanism for bone regulation. Megakaryocytes (MKs) are one such cell type that potentially acts as a master regulator of the bone marrow microenvironment due to its effects on hematopoiesis, osteoblastogenesis, and osteoclastogenesis. While several of these processes are induced/inhibited through MK secreted factors, others are primarily regulated by direct cell-cell contact. Notably, the regulatory effects that MKs exert on these different cell populations has been found to change with aging and disease states. Overall, MKs are a critical component of the bone marrow that should be considered when examining regulation of the skeletal microenvironment. An increased understanding of the role of MKs in these physiological processes may provide insight into novel therapies that can be used to target specific pathways important in hematopoietic and skeletal disorders.

Autologous Collagen-Induced Chondrogenesis: From Bench to Clinical Development.

Microfracture is a common technique that uses bone marrow components to stimulate cartilage regeneration. However, the clinical results of microfracture range from poor to good. To enhance cartilage healing, several reinforcing techniques have been developed, including porcine-derived collagen scaffold, hyaluronic acid, and chitosan. Autologous collagen-induced chondrogenesis (ACIC) is a single-step surgical technique for cartilage regeneration that combines gel-type atelocollagen scaffolding with microfracture. Even though ACIC is a relatively new technique, literature show excellent clinical results. In addition, all procedures of ACIC are performed arthroscopically, which is increasing in preference among surgeons and patients. The ACIC technique also is called the Shetty-Kim technique because it was developed from the works of A.A. Shetty and S.J. Kim. This is an up-to-date review of the history of ACIC.

StemRegenin 1 Mitigates Radiation-Mediated Hematopoietic Injury by Modulating Radioresponse of Hematopoietic Stem/Progenitor Cells

Hematopoietic injury resulting from the damage of hematopoietic stem/progenitor cells (HSPCs) can be induced by either nuclear accident or radiotherapy. Radiomitigation of HSPCs is critical for the development of medical countermeasure agents. StemRegenin 1 (SR1) modulates the maintenance and function of HSPCs under non-stress conditions. However, the impact of SR1 in radiation-induced hematopoietic injury both in vivo and in vitro remains unknown. In this study, we found that treatment with SR1 after irradiation of C57BL/6 mice significantly mitigates TBI-induced death (80% of SR1-treated mice survival vs. 30% of saline-treated mice survival) with enhanced recovery of peripheral blood cell counts, with the density and cell proliferation of bone marrow components as observed by Hematoxylin and Eosin (H&E) and Ki-67 staining. Interestingly, in vitro analysis of human HSPCs showed that SR1 enhanced the population of human HSPCs (CD34+) under both non-irradiating and irradiating conditions, and reduced radiation-induced DNA damage and apoptosis. Furthermore, SR1 attenuated the radiation-induced expression of a member of the pro-apoptotic BCL-2 family and activity of caspase-3. Overall, these results suggested that SR1 modulates the radioresponse of HSPCs and might provide a potential radiomitigator of hematopoietic injury, which contributes to increase the survival of patients upon irradiation.

Endothelial and Leptin Receptor+ cells promote the maintenance of stem cells and hematopoiesis in early postnatal murine bone marrow.

Mammalian hematopoietic stem cells (HSCs) colonize the bone marrow during late fetal development, and this becomes the major site of hematopoiesis after birth. However, little is known about the early postnatal bone marrow niche. We performed single-cell RNA sequencing of mouse bone marrow stromal cells at 4days, 14days, and 8weeks after birth. Leptin-receptor-expressing (LepR+) stromal cells and endothelial cells increased in frequency during this period and changed their properties. At all postnatal stages, LepR+ cells and endothelial cells expressed the highest stem cell factor (Scf) levels in the bone marrow. LepR+ cells expressed the highest Cxcl12 levels. In early postnatal bone marrow, SCF from LepR+/Prx1+ stromal cells promoted myeloid and erythroid progenitor maintenance, while SCF from endothelial cells promoted HSC maintenance. Membrane-bound SCF in endothelial cells contributed to HSC maintenance. LepR+ cells and endothelial cells are thus important niche components in early postnatal bone marrow.

Mesenchymal Stem Cells in Acquired Aplastic Anemia: The Spectrum from Basic to Clinical Utility

Aplastic anemia (AA), a rare but potentially life-threatening disease, is a paradigm of bone marrow failure syndromes characterized by pancytopenia in the peripheral blood and hypocellularity in the bone marrow. The pathophysiology of acquired idiopathic AA is quite complex. Mesenchymal stem cells (MSCs), an important component of the bone marrow, are crucial in providing the specialized microenvironment for hematopoiesis. MSC dysfunction may result in an insufficient bone marrow and may be associated with the development of AA. In this comprehensive review, we summarized the current understanding about the involvement of MSCs in the pathogenesis of acquired idiopathic AA, along with the clinical application of MSCs for patients with the disease. The pathophysiology of AA, the major properties of MSCs, and results of MSC therapy in preclinical animal models of AA are also described. Several important issues regarding the clinical use of MSCs are discussed finally. With evolving knowledge from basic studies and clinical applications, we anticipate that more patients with the disease can benefit from the therapeutic effects of MSCs in the near future.

Panchkarma in Janpadodhwansa w.s.r. COVID-19 and Its Documentation from Ayurvedic Texts

COVID-19 has emerged as a public health crisis and has done caused a disaster at multiple -levels. Manual retrieval of ancient books of Ayurveda was carried out. In From an Ayurvedic point of view, its very difficult to correlate COVID-19 with Aayurvedic terminology, on the basis of causes, symptoms, and complications., Iit can be correlated with various diseases like Janpadodhwansha(~epidemic), Shwasa(respiratory disease), pranaavrit vata(subtype of vata that is seated in head), dushi-visha(~cumulative), jwara-sannipattika (~severe fever), vata-kaphaja jwar (fever) , shwasana (~pneumonia), Abhisangaj jwar ( ~fever caused by virus or evil spirit), majjadhatugata jwar (~vitiated regulatory factors are located in structural component of bone marrow ) &and kasa roga (coughing). Treatment in such conditions is mentioned as Panchkarma (~detoxification procedure) . The main goal in such type of condition is to remove avarana (overlapping) so that Vayu (air) returns to normal functioning. Five detoxification procedures were also reported to be effective against a broad spectrum of viruses, including human immunodeficiency virus, herpes viruses, etc. Ayurvedic detoxification procedures can add an additive effect in to the managing COVID-19 through multiple modules, like as prophylactic and, curative treatment. Implementation of suggested influence or mode of action probably come up with empirical discernment escalating the Ayurveda other than prophylactic medical management and system considering alike ailments.

An Audit of the Length of Bone Marrow Trephine Biopsy in Adult Patients: A Cross-sectional Study

Introduction: A bone marrow trephine biopsy is a minor surgical procedure used to diagnose various haematological and non haematological diseases, such as leukaemia, multiple myeloma, and parasitic disorders like histoplasmosis and leishmaniasis. It is often performed alongside bone marrow aspiration to gather additional information about cellularity, the arrangement of marrow components, and the presence of focal diseases. According to the World Health Organisation (WHO), the recommended minimum adequate length for a trephine biopsy is ≥1.5 cm (before processing). Aim: To conduct an audit of trephine biopsy lengths and assess their diagnostic utility among adult patients. Materials and Methods: This cross-sectional study analysed all bone marrow biopsies from adult patients received at the Department of Pathology, Christian Medical College and Hospital in Ludhiana, Punjab, India, over a three-year period (January 2019 to December 2021). The biopsies were categorised into three groups based on their length at the time of grossing: Group A (≥1.5 cm), Group B (1-1.4 cm), and Group C (0.5-0.9 cm). The length of each trephine biopsy was recorded and its diagnostic usefulness was evaluated. Results: The study included 1,155 trephine biopsies performed over the three-year period. Out of these, 1042, 97, and 16 biopsies were allocated to Groups A, B, and C, respectively. Biopsies meeting the recommended length (>1.5 cm) accounted for 90.2% of the cases. Longer biopsies were associated with a higher rate of conclusive diagnosis compared to shorter biopsies (p-value=0.02). However, when comparing Groups A and B individually, no significant difference was observed in terms of the conclusive diagnosis (p-value=0.9). Conclusion: Trephine biopsy is a vital tool for diagnosing haematological disorders, particularly focal lesions. Obtaining longer trephine biopsies should be emphasised, as they contribute to a more definitive diagnosis.

THE MORPHOLOGICAL AND FUNCTIONAL STATE OF THE VASCULAR COMPONENT OF HEMATOPOIESIS ORGANS IN DIFFERENT TIMES OF SIMULATED HYPOXIA IN THE EXPERIMENT

Under conditions of hypoxia, a change in the morphological and functional state of the components of the microvasculature bed will be accompanied by a change in the adequacy and usefulness of the immune response and hematopoiesis. The purpose of the study was to determine the morphofunctional state of the vascular component of the red bone marrow and spleen at various times of simulated hypoxia and to identify the degree of influence of CD68-positive cells on this state. Chronic hypoxia was modeled on 246 outbred male rats using special chambers containing natural gas from the Astrakhan gas field (Russia) at a concentration not exceeding the maximum allowable. The experiment was continued for 120 days, inhalation was carried out five days a week for four hours a day, the removal of animals from the experiment was carried out every 30 days. The functional activity of the vascular component of the red bone marrow was determined using the method of laser Doppler flowmetry. The degree of expression of inducible (iNOS) and endothelial (eNOS) NO synthases and the distribution of CD68-positive cells in the spleen structures were determined by immunohistochemical method. The study showed that as the duration of chronic simulated hypoxia increases, there is a decrease in the microcirculation index, an increase in myogenic tone and shunting index, which, taken together, indicates a deterioration of organ perfusion and confirms the formation of a hypoxic state. An analysis of the functional activity, carried out using an immunohistochemical study of the expression of iNOS and eNOS in the structures of the spleen, showed that as the duration of the experiment increased, the level of endothelial synthase decreased and the level of inducible synthase increased. Perhaps this is due to the influence of biologically active substances secreted by macrophages activated during hypoxia. This is supported by an increase in the presence of CD68-positive cells in the red pulp and along the connective tissue trabeculae as the experimental exposure increases.

Raman microspectroscopy reveals unsaturation heterogeneity at the lipid droplet level and validates an in vitro model of bone marrow adipocyte subtypes.

Bone marrow adipocytes (BMAds) constitute the most abundant stromal component of adult human bone marrow. Two subtypes of BMAds have been described, the more labile regulated adipocytes (rBMAds) and the more stable constitutive adipocytes (cBMAds), which develop earlier in life and are more resilient to environmental and metabolic disruptions. In vivo, rBMAds are enriched in saturated fatty acids, contain smaller lipid droplets (LDs) and more readily provide hematopoietic support than their cBMAd counterparts. Mouse models have been used for BMAds research, but isolation of primary BMAds presents many challenges, and thus in vitro models remain the current standard to study nuances of adipocyte differentiation. No in vitro model has yet been described for the study of rBMAds/cBMAds. Here, we present an in vitro model of BM adipogenesis with differential rBMAd and cBMAd-like characteristics. We used OP9 BM stromal cells derived from a (C57BL/6xC3H)F2-op/op mouse, which have been extensively characterized as feeder layer for hematopoiesis research. We observed similar canonical adipogenesis transcriptional signatures for spontaneously-differentiated (sOP9) and induced (iOP9) cultures, while fatty acid composition and desaturase expression of Scd1 and Fads2 differed at the population level. To resolve differences at the single adipocyte level we tested Raman microspectroscopy and show it constitutes a high-resolution method for studying adipogenesis in vitro in a label-free manner, with resolution to individual LDs. We found sOP9 adipocytes have lower unsaturation ratios, smaller LDs and higher hematopoietic support than iOP9 adipocytes, thus functionally resembling rBMAds, while iOP9 more closely resembled cBMAds. Validation in human primary samples confirmed a higher unsaturation ratio for lipids extracted from stable cBMAd-rich sites (femoral head upon hip-replacement surgery) versus labile rBMAds (iliac crest after chemotherapy). As a result, the 16:1/16:0 fatty acid unsaturation ratio, which was already shown to discriminate BMAd subtypes in rabbit and rat marrow, was validated to discriminate cBMAds from rBMAd in both the OP9 model in vitro system and in human samples. We expect our model will be useful for cBMAd and rBMAd studies, particularly where isolation of primary BMAds is a limiting step.

Imbalanced M1 and M2 Macrophage Polarization in Bone Marrow Provokes Impairment of the Hematopoietic Microenvironment in a Mouse Model of Hemophagocytic Lymphohistiocytosis.

Lipopolysaccharide (LPS) treatment induced hemophagocytic lymphohistiocytosis in senescence-accelerated mice (SAMP1/TA-1), but not in senescence-resistant control mice (SAMR1). SAMP1/TA-1 treated with LPS exhibited functional impairment of the hematopoietic microenvironment, which disrupted the dynamics of hematopoiesis. Macrophages are a major component of the bone marrow (BM) hematopoietic microenvironment, which regulates hematopoiesis. Qualitative and quantitative changes in activated macrophages in LPS-treated SAMP1/TA-1 are thought to contribute to the functional deterioration of the hematopoietic microenvironment. Thus, we examined the polarization of pro-inflammatory (M1) and anti-inflammatory (M2) macrophages, and the dynamics of macrophage production in the BM of SAMP1/TA-1 and SAMR1 after LPS treatment. After LPS treatment, the proportions of M1 and M2 macrophages and the numbers of macrophage progenitor (CFU-M) cells increased in both SAMP1/TA-1 and SAMR1. However, compared to the SAMR1, the increase in the M1 macrophage proportion was prolonged, and the increase in the M2 macrophage proportion was delayed. The increase in the number of CFU-M cells was prolonged in SAMP1/TA-1 after LPS treatment. In addition, the levels of transcripts encoding an M1 macrophage-inducing cytokine (interferon-γ) and macrophage colony-stimulating factor were markedly increased, and the increases in the levels of transcripts encoding M2 macrophage-inducing cytokines (interleukin (IL)-4, IL-10, and IL-13) were delayed in SAMP1/TA-1 when compared to SAMR1. Our results suggest that LPS treatment led to the severely imbalanced polarization of activated M1/M2 macrophages accompanied by a prolonged increase in macrophage production in the BM of SAMP1/TA-1, which led to the impairment of the hematopoietic microenvironment, and disrupted the dynamics of hematopoiesis.