Stromal Precursor Research Articles

Adipose-tissue Treg cells restrain differentiation of stromal adipocyte precursors to promote insulin sensitivity and metabolic homeostasis

Regulatory T (Treg) cells in epidydimal visceral adipose tissue (eVAT) of lean mice and humans regulate metabolic homeostasis. We found that constitutive or punctual depletion of eVAT-Treg cells reined in the differentiation of stromal adipocyte precursors. Co-culture of these precursors with conditional medium from eVAT-Treg cells limited their differentiation invitro, suggesting a direct effect. Transcriptional comparison of adipocyte precursors, matured in the presence or absence of the eVAT-Treg-conditioned medium, identified the oncostatin-M (OSM) signaling pathway as a key distinction. Addition of OSM to invitro cultures blocked the differentiation of adipocyte precursors, while co-addition of anti-OSM antibodies reversed the ability of the eVAT-Treg-conditioned medium to inhibit invitro adipogenesis. Genetic depletion of OSM (specifically in Treg) cells or of the OSM receptor (specifically on stromal cells) strongly impaired insulin sensitivity and related metabolic indices. Thus, Treg-cell-mediated control of local progenitor cells maintains adipose tissue and metabolic homeostasis, a regulatory axis seemingly conserved in humans.

Linoleic acid blunts early osteoblast differentiation and impairs oxidative phosphorylation in vitro

BackgroundLinoleic acid (LNA), an essential polyunsaturated fatty acid (PUFA), plays a crucial role in cellular functions. However, excessive intake of LNA, characteristic of Western diets, can have detrimental effects on cells and organs. Human observational studies have shown an inverse relationship between plasma LNA concentrations and bone mineral density. The mechanism by which LNA impairs the skeleton is unclear, and there is a paucity of research on the effects of LNA on bone-forming osteoblasts. MethodsThe effect of LNA on osteoblast differentiation, cellular bioenergetics, and production of oxidized PUFA metabolites in vitro, was studied using primary mouse bone marrow stromal cells (BMSC) and MC3T3-E1 osteoblast precursors. ResultsLNA treatment decreased alkaline phosphatase activity, an early marker of osteoblast differentiation, but had no effect on committed osteoblasts or on mineralization by differentiated osteoblasts. LNA suppressed osteoblast commitment by blunting the expression of Runx2 and Osterix, key transcription factors involved in osteoblast differentiation, and other key osteoblast-related factors involved in bone formation. LNA treatment was associated with increased production of oxidized LNA- and arachidonic acid-derived metabolites and blunted oxidative phosphorylation, resulting in decreased ATP production. ConclusionOur results show that LNA inhibited early differentiation of osteoblasts and this inhibitory effect was associated with increased production of oxidized PUFA metabolites that likely impaired energy production via oxidative phosphorylation.

ДИФФЕРЕНЦИАЛЬНАЯ ЭКСПРЕССИЯ ГЕНОВ МОЛЕКУЛ АДГЕЗИИ И МЕЖКЛЕТОЧНОГО ВЗАИМОДЕЙСТВИЯ В МИКРОЭКСПЛАНТАХ КОСТНОГО МОЗГА ПРИ МОДЕЛИРОВАНИИ МИКРОГРАВИТАЦИИ

Functional activity of the bone marrow cells is dependent on external signals received from a local microenvironment. In addition to the soluble growth factors and hormones, contacts with neighbor cells and signals from the extracellular matrix are also the most important homeostasis regulators in the bone marrow niche. Purpose of the work is to explore the effects of simulated microgravity on the transcription activity of genes the products of which define the cell-to-cell and cell-to-matrix interactions in bone marrow micro-explants. Femoral marrow cells from male mice BalbC 19-20 wks of age were used in the investigation. After isolation, bone marrow cells were pooled and exposed to simulated microgravity in vitro over 14 days. Following the exposure, bone marrow micro-explants were found to increase expression of genes of the cell adhesion molecules responsible for cell-to-cell interaction (Ncam1, Icam1, Vcam1), and integrins α-М (Itgam) and β-1 (Itgb1). Also, there was a reliable decrease in expression of genes involved in the cell-to-matrix interaction, including integrins β-3 (Itgb3), β-2 (Itgb2), α-Х (Itgax), α-l (Itga1), α-5 (Itga5), receptor CD44 (Cd44), and molecules of the adhesion complexes, such as α- and β-katenins (Ctnna1, Ctnnb1). These changes in the pattern of cells-to-environment interaction may induce disorders in self-renewal, proliferation and differentiation of stromal and hemopoietic precursors which may end up with distortion of hemopoiesis, change in bone homeostasis leading to osteopenia observed after space missions.

Microgravity effects and aging physiology: similar changes or common mechanisms?

Despite the use of preventive measures (including intense physical activity), cosmonauts and astronauts develop muscle atony and atrophy, insufficiency of the cardiovascular system, osteopenia, etc. All these changes, reminiscent of age-related physiological changes, occur in a healthy person in microgravity quite quickly - within a few months. Adaptation to the absence of gravity leads to the symptoms of aging, which are compensated after returning to Earth. The prospect of interplanetary flights raises the question of gravity thresholds, below which the main physiological systems will lose their functional potential, similar to aging, and affect life expectancy. An important role in the aging process belongs to the body’s cellular reserve - progenitor cells, which are involved in physiological remodeling and regenerative/reparative processes of all physiological systems. With age, progenitor cell count and the regenerative potential decreases. Moreover, their paracrine spectrum becomes pro-inflammatory during replicative senescence, disrupting tissue homeostasis. Mesenchymal stem/stromal cells (MSCs) are mechanosensitive, and therefore the absence of a gravitational stimulus causes serious changes in their functional status. The review compares the cellular effects of microgravity and changes developing in senescent cells, including stromal precursors.

Stromal Lineage Precursors from Rodent Femur and Tibia Bone Marrows after Hindlimb Unloading: Functional Ex Vivo Analysis.

Rodent hindlimb unloading (HU) model was developed to elucidate responses/mechanisms of adverse consequences of space weightlessness. Multipotent mesenchymal stromal cells (MMSCs) were isolated from rat femur and tibia bone marrows and examined ex vivo after 2 weeks of HU and subsequent 2 weeks of restoration of load (HU + RL). In both bones, decrease of fibroblast colony forming units (CFU-f) after HU with restoration after HU + RL detected. In CFU-f and MMSCs, levels of spontaneous/induced osteocommitment were similar. MMSCs from tibia initially had greater spontaneous mineralization of extracellular matrix but were less sensitive to osteoinduction. There was no recovery of initial levels of mineralization in MMSCs from both bones during HU + RL. After HU, most bone-related genes were downregulated in tibia or femur MMSCs. After HU + RL, the initial level of transcription was restored in femur, while downregulation persisted in tibia MMSCs. Therefore, HU provoked a decrease of osteogenic activity of BM stromal precursors at transcriptomic and functional levels. Despite unidirectionality of changes, the negative effects of HU were more pronounced in stromal precursors from distal limb-tibia. These observations appear to be on demand for elucidation of mechanisms of skeletal disorders in astronauts in prospect of long-term space missions.

Epigenetic regulation of cancer-associated fibroblast heterogeneity.

Cancer-associated fibroblasts (CAFs), a significant component of the tumor microenvironment (TME), contribute to cancer progression through the secretion of extracellular matrix (ECM), growth factors, and metabolites. It is now well recognized that CAFs are a heterogenous population with ablation experiments leading to reduced tumor growth and single-cell RNA sequencing demonstrating CAF subgroups. CAFs lack genetic mutations yet substantially differ from their normal stromal precursors. Here, we review epigenetic changes in CAF maturation, focusing on DNA methylation and histone modifications. DNA methylation changes in CAFs have been demonstrated globally, while roles of methylation at specific genes affect tumor growth. Further, loss of CAF histone methylation and gain of histone acetylation has been shown to promote CAF activation and tumor promotion. Many CAF activating factors, such as transforming growth factor β (TGFβ), lead to these epigenetic changes. MicroRNAs (miRNAs) serve as targets and orchestrators of epigenetic modifications that influence gene expression. Bromodomain and extra-terminal domain (BET), an epigenetic reader, recognizes histone acetylation and activates the transcription of genes leading to the pro-tumor phenotype of CAFs.

IL-25 directly modulates adipocyte function and inflammation through the regulation of adiponectin.

This study aimed to investigate the direct role of IL-25 in modulating adipocyte function during homeostasis and low-grade inflammation induced by lipopolysaccharide (LPS). The 3T3-L1 preadipocyte cell lines and primary cultures of adipose-derived stromal vascular precursor cells of wild-type and IL-17RB-deficient mice were used to determine the direct function of IL-25. The expression of IL-17RB in differentiating adipocyte was determined using real-time PCR and flow cytometry analysis. The effect of IL-25 on lipid accumulation, triglyceride content, lipolysis, glucose uptake, and adipokine expression in the mature adipocytes was evaluated. IL-25 modulating the expression of inflammatory cytokines in adipocytes induced by low dose LPS was determined using real-time PCR and ELISA. The receptor for IL-25 was up-regulated during adipocyte differentiation and IL-25 directly modulated adipocyte function by reducing lipid accumulation and triglyceride concentration and enhancing lipolysis without affecting an insulin-stimulated glucose uptake. Interestingly, IL-25 induced adiponectin secretion through the PI3K/AKT signaling pathway. In 3T3-L1 adipocytes under low-grade inflammation, IL-25 attenuated the expression of IL-6 and CCL5 through the induction of adiponectin. Our studies suggest that IL-25 directly regulates adipocyte function by maintaining the adiponectin level during homeostasis and by alleviating inflammatory response through the regulation of adiponectin during low-grade inflammation in adipocytes.

Fate mapping and scRNA sequencing reveal origin and diversity of lymph node stromal precursors

Lymph node (LN) stromal cells play a crucial role in LN development and in supporting adaptive immune responses. However, their origin, differentiation pathways, and transcriptional programs are still elusive. Here, we used lineage-tracing approaches and single-cell transcriptome analyses to determine origin, transcriptional profile, and composition of LN stromal and endothelial progenitors. Our results showed that all major stromal cell subsets and a large proportion of blood endothelial cells originate from embryonic Hoxb6+ progenitors of the lateral plate mesoderm (LPM), whereas lymphatic endothelial cells arise from Pax3+ progenitors of the paraxial mesoderm (PXM). Single-cell RNA sequencing revealed the existence of different Cd34+ and Cxcl13+ stromal cell subsets and showed that embryonic LNs contain proliferating progenitors possibly representing the amplifying populations for terminally differentiated cells. Taken together, our work identifies the earliest embryonic sources of LN stromal and endothelial cells and demonstrates that stromal diversity begins already during LN development.

Identification, Isolation, and Characterization of Melanocyte Precursor Cells in the Human Limbal Stroma.

Given their vital role in the homeostasis of the limbal stem cell niche, limbal melanocytes have emerged as promising candidates for tissue engineering applications. This study aimed to isolate and characterize a population of melanocyte precursors in the limbal stroma, compared with melanocytes originating from the limbal epithelium, using magnetic-activated cell sorting (MACS) with positive (CD117/c-Kit microbeads) or negative (CD326/EpCAM or anti-fibroblast microbeads) selection approaches. Both approaches enabled fast and easy isolation and cultivation of pure limbal epithelial and stromal melanocyte populations, which differed in phenotype and gene expression, but exhibited similar functional properties regarding proliferative potential, pigmentation, and support of clonal growth of limbal epithelial stem/progenitor cells (LEPCs). In both melanocyte populations, limbus-specific matrix (laminin 511-E8) and soluble factors (LEPC-derived conditioned medium) stimulated melanocyte adhesion, dendrite formation, melanogenesis, and expression of genes involved in UV protection and immune regulation. The findings provided not only a novel protocol for the enrichment of pure melanocyte populations from limbal tissue applying easy-to-use MACS technology, but also identified a population of stromal melanocyte precursors, which may serve as a reservoir for the replacement of damaged epithelial melanocytes and an alternative resource for tissue engineering applications.

Short-Term Reloading After Prolonged Unloading Ensures Restoration of Stromal but Not Hematopoietic Precursor Activity in Tibia Bone Marrow of C57Bl/6N Mice.

Bone and muscle tissues are mostly susceptible to different kinds of hypodynamia, including real and simulated microgravity (sμg). To evaluate the effect of sμg on bone marrow (BM), male C57Bl/6N mice were divided into three groups: vivarium control (VC), 30-day hindlimb suspension (HS), and subsequent 12-h short-term support reloading (RL). The effects on BM total mononucleated cells (MNCs) as well as stromal and hematopoietic progenitors from murine tibia were studied. The number of BM MNCs, immunophenotype, proliferation, colony-forming units (CFUs), differentiation and secretory activity of hematopoietic and stromal BM cells were determined. HS led to a twofold decrease in MNCs, alteration of surface molecule expression profiles, suppression of proliferative activity of BM cells, and change of soluble mediators' levels. The stromal compartment was characterized by a decrease of CFU of fibroblasts and suppression of spontaneous osteo-commitment after HS. Among the hematopoietic precursors, a decrease in the total number of CFUs was found mainly at the expense of suppression of CFU-GM and CFU-GEMM. After RL, restoration of the stromal precursor's functional activity to control levels and overabundance of paracrine mediator's production were detected, whereas the complete recovery of hematopoietic precursor's activity did not occur. These data demonstrate the fast functional reaction of the stromal compartment on restoration of loading support.

Angiogenin Released from ABCB5+ Stromal Precursors Improves Healing of Diabetic Wounds by Promoting Angiogenesis

Severe angiopathy is a major driver for diabetes-associated secondary complications. Knowledge on the underlying mechanisms essential for advanced therapies to attenuate these pathologies is limited. Injection of ABCB5+ stromal precursors at the edge of nonhealing diabetic wounds in a murine db/db model, closely mirroring human type 2 diabetes, profoundly accelerates wound closure. Strikingly, enhanced angiogenesis was substantially enforced by the release of the ribonuclease angiogenin from ABCB5+ stromal precursors. This compensates for the profoundly reduced angiogenin expression in nontreated murine chronic diabetic wounds. Silencing of angiogenin in ABCB5+ stromal precursors before injection significantly reduced angiogenesis and delayed wound closure in diabetic db/db mice, implying an unprecedented key role for angiogenin in tissue regeneration in diabetes. These data hold significant promise for further refining stromal precursors-based therapies of nonhealing diabetic foot ulcers and other pathologies with impaired angiogenesis.

Development of follicular dendritic cells in lymph nodes depends on retinoic acid-mediated signaling.

Specialized stromal cells occupy and help define B- and T-cell domains, which are crucial for proper functioning of our immune system. Signaling through lymphotoxin and TNF receptors is crucial for the development of different stromal subsets, which are thought to arise from a common precursor. However, mechanisms that control the selective generation of the different stromal phenotypes are not known. Using in vitro cultures of embryonic mouse stromal cells, we show that retinoic acid-mediated signaling is important for the differentiation of precursors towards the Cxcl13pos follicular dendritic cell (FDC) lineage, and also blocks lymphotoxin-mediated Ccl19pos fibroblastic reticular cell lineage differentiation. Accordingly, at the day of birth we observe the presence of Cxcl13posCcl19neg/low and Cxcl13neg/lowCcl19pos cells within neonatal lymph nodes. Furthermore, ablation of retinoic acid receptor signaling in stromal precursors early after birth reduces Cxcl13 expression, and complete blockade of retinoic acid signaling prevents the formation of FDC networks in lymph nodes.

SHP-2 deletion in CD4Cre expressing chondrocyte precursors leads to tumor development with wrist tropism

Due to redundancy with other tyrosine phosphatases, the ubiquitously expressed tyrosine phosphatase SHP-2 (encoded by Ptpn11) is not required for T cell development. However, Ptpn11 gene deletion driven by CD4 Cre recombinase leads to cartilage tumors in the wrist. Using a fate mapping system, we demonstrate that wrist tumor development correlates with increased frequency and numbers of non-hematopoietic lineage negative CD45 negative cells with a bone chondrocyte stromal cell precursor cell (BCSP) phenotype. Importantly, the BCSP subset has a history of CD4 expression and a marked wrist location tropism, explaining why the wrist is the main site of tumor development. Mechanistically, we found that in SHP-2 absence, SOX-9 is no longer regulated, leading to an uncontrolled proliferation of the BCSP subset. Altogether, these results identify a unique subset of chondrocyte precursors tightly regulated by SHP-2. These findings underscore the need for the development of methods to therapeutically target this subset of cells, which could potentially have an impact on treatment of SHP-2 dysfunction linked debilitating diseases.

Lung-Resident Mesenchymal Stem Cell Fates within Lung Cancer

Simple SummaryLung cancer remains the leading cause of cancer-related deaths worldwide. Herein, the heterogeneous tumor stroma decisively impacts on tumor progression, therapy resistance, and, thus, poor clinical outcome. Among the numerous non-epithelial cells constructing the complex environment of lung carcinomas, mesenchymal stem cells (MSC) gained attraction being stromal precursor cells that could be recruited and ‘educated’ by lung cancer cells to adopt a tumor-associated MSC phenotype, serve as source for activated fibroblasts and presumably for vascular mural cells finally reinforcing tumor progression. Lung-resident MSCs should be considered as ‘local MSCs in stand by’ ready to be arranged within the cancer stroma.Lung-resident mesenchymal stem cells (LR-MSCs) are non-hematopoietic multipotent stromal cells that predominately reside adventitial within lung blood vessels. Based on their self-renewal and differentiation properties, LR-MSCs turned out to be important regulators of normal lung homeostasis. LR-MSCs exert beneficial effects mainly by local secretion of various growth factors and cytokines that in turn foster pulmonary regeneration including suppression of inflammation. At the same time, MSCs derived from various tissues of origins represent the first choice of cells for cell-based therapeutic applications in clinical medicine. Particularly for various acute as well as chronic lung diseases, the therapeutic applications of exogenous MSCs were shown to mediate beneficial effects, hereby improving lung function and survival. In contrast, endogenous MSCs of normal lungs seem not to be sufficient for lung tissue protection or repair following a pathological trigger; LR-MSCs could even contribute to initiation and/or progression of lung diseases, particularly lung cancer because of their inherent tropism to migrate towards primary tumors and metastatic sites. However, the role of endogenous LR-MSCs to be multipotent tumor-associated (stromal) precursors remains to be unraveled. Here, we summarize the recent knowledge how ‘cancer-educated’ LR-MSCs impact on lung cancer with a focus on mesenchymal stem cell fates.

Intraosseous Administration of Multipotent Mesenchymal Stromal Cells to Patients with Graft Failure after Allogeneic Hematopoietic Stem Cell Transplantation

IntroductionMultipotent mesenchymal stromal cells (MSCs) are involved in the formation of niches for hematopoietic stem cells and support hematopoiesis.There are several reasons for the graft failure in patients after allogeneic hematopoietic stem cell transplantation (allo-HSCT). One of them could be the damage of hematopoietic microenvironment by chemotherapeutic drugs and tumor cells. As a result, the bone marrow (BM) stroma loses its ability to support hematopoiesis.Perhaps the donor's MSCs can serve as a source of hematopoietic niches recovery in these cases.The aim of the study was to investigate the possibility to restore the hematopoietic stromal microenvironment in patients with graft failure with donors MSCs implanted into trabecular bone.Patients and methodsA randomized study for acute graft versus host disease prophylaxis with MSCs has been conducted by the National Research Center for Hematology, Moscow, Russia. MSCs were developed for each patient from their individual BM donor and cryopreserved.There was long-term BM aplasia observed in 4 patients with GF following allo-HSCT. All of the patients received a second allo-HSCT with MSCs from the donor BM. The MSCs were injected under local anesthesia in the iliac crest that is, directly into the bone marrow in the trabecular bone area. All patients provided informed consent. The implanted MSCs from several passages were thawed and washed free of dimethyl sulfoxide. The cells were then suspended in 2 ml of 6% polyglucin. The cells (200-370 х106) were injected into the bone tissue in small portions (100-200 microliters) through 2 skin and multiple periosteal punctures.BM punctures were performed for patients 1, 2 and 4.5 months after donor MSC implantation simultaneously. BM (2-3 ml) was collected from 2-4 independent punctures of the iliac spine for each patient. From these BM samples MSCs were isolated and colony forming unit fibroblast (CFU-F) were analyzed according to standard protocols.The proportion of chimeric DNA was determined in the patients' MSCs at the 1-3rd passages and CFU-F. The cell DNA was isolated by standard methods of extraction and salt precipitation with isopropanol. The chimerism in MSCs was analyzed by the STR-PCR method. The protocol uses reagents for the multiplex analysis of 19 STR-markers and the human amelogenin locus. The informative loci were preselected to monitor the chimerism in the observed patients.ResultsIn all 4 cases, after intraosseous injection of MSCs and re-allo-HSCT from the same donor, the patients had a restoration of donor hematopoiesis.In stromal precursor cells - MSCs and CFU-F, obtained from the BM of patients at 1, 2 and 4.5 months after the implantation of MSCs and second allo-HSCT from the same donor, donors' markers were identified. The table presents the data on chimerism in the BM and studied MSCs and CFU-F in patients.The sites of BM punctures in patients only approximately coincided with the place of injection of donor MSCs, therefore the proportion of donor cells does not clearly reflect the content of donor stromal cells in patients. Nevertheless, it can be argued that implanted MSCs remain localized at the site of administration and do not lose the ability to proliferate. In fact, these cells survived 3 transfers: from the donor's organism to the culture, then again into the patient's body and repeatedly into the culture. The presence of donor CFU-F - descendants of MSCs proves the functional activity of these cells.ConclusionsIt can be assumed that MSCs caused the restoration of donor hematopoiesis, they probably performed a trophic function, participated in the restoration of niches for hematopoietic cells, or had an immunomodulating effect preventing re-failure of the hematopoietic cell transplant.Thus, the long-term existence of donor's MSCs in the patient's organism is shown for the first time. A strong potential of MSCs for repeated transfers was revealed. It has been shown that possibly intraosseous MSCs implantation could facilitate transplant engraftment.The materials are supported by grant from the Russian Science Foundation, Project № 16-15-00102 [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

The Tumor Microenvironment in Follicular Lymphoma: Its Pro-Malignancy Role with Therapeutic Potential.

In the follicular lymphoma (FL) microenvironment, CXCR5+ICOS+PD1+BCL6+ follicular helper T (Tfh) cells, which closely correlate with FL B cells in neoplastic follicles, play a major role in supporting FL. Interleukin-4 secreted by Tfh cells triggers the upregulation of the lymphocyte chemoattractant CXCL12 in stromal cell precursors, in particular by fibroblastic reticular cells (FRCs). In turn, mesenchymal stem cells (MSCs) can be committed to FRC differentiation in the bone marrow and lymph nodes involved by FL. Noteworthy, MSCs can promote the differentiation of Tfh cells into highly immunosuppressive T-follicular regulatory cells. The tumor suppressor HVEM is highly mutated in FL cells, and its deficiency increases Tfh cell frequency. In contrast, PI3Kδ inhibition impedes the recruitment of Tfh/regulatory T cells and impairs the proliferation of follicular dendritic cells (FDCs) and FDC-induced angiogenesis. Since TIGIT ligands are expressed by FDCs, the immune checkpoint receptor TIGIT plays an important role in tumor-infiltrating T cells. Thus, TIGIT blockade might invigorate cytotoxic T cells in the FL microenvironment. Given their potential to simultaneously reduce the neoplastic B cells, Tfh, and TFR cells could also reinforce the effects of the cytotoxic T cells. This combinatory strategy should be explored as a treatment option to tackle FL.

Stromal Precursors in the Bone Marrow of Untreated Patients with Severe and Non-Severe Aplastic Anemia Differ in the Proliferative Potential

Background. Aplastic anemia (AA) is a disorder characterized by pancytopenia, hypoplastic bone marrow (BM), and the absence of underlying malignancy. It is believed to be of autoimmune nature. However, some patients fail to respond to the immunosuppressive therapy. The impaired hematopoietic microenvironment could be another reason for BM failure. The severity of AA varies widely from mild, chronic pancytopenia to total hematopoietic failure. The diagnosis of severe (SAA) and non-severe AA (NAA) is based on an absolute neutrophil count as an essential criterion. The aim of the study was to analyze the multipotent mesenchymal stromal cells (MMSC) and fibroblasts colony forming units (CFU-F) in BM of untreated SAA and NAA patients. Methods. The study included 22 AA patients (8 with SAA and 14 with NAA) in the debut of the disease. In all patients BM was aspirated after informed consent at diagnostic punctures. The proportion of non-hematopoietic CD45-CD34-CD71-CD235-CD90+CD73+CD105+ cells was estimated by FACS. From the BM, MMSC were isolated by the standard method and the concentration of CFU-F was determined. Individual CFU-F-derived colonies were analyzed for their proliferative and differentiation potential. Adipogenic and osteogenic differentiation potential was analyzed with standard techniques. Relative expression level (REL) of several genes had been estimated with RT-PCR in Taqman modification. As a control 19 BM samples of healthy donors of according age were used. Results. The data are presented in the table. The proportion of non-hematopoietic cells was higher in the BM of AA patients than in healthy donors. We recalculated the proportion of CFU-F among non- hematopoietic cells; it was similar in the BM of AA patients and healthy donors. However, the concentration of -CFU-F was much higher in the BM of patients with SAA then in the BM of patients with NAA. Among NAA patients, 2 had PNG clone and unlike other NAA patients increased CFU-F concentration, comparable to patients with SAA. It seems that the character of stromal cell damage depends on the severity of AA. Individual CFU-F- derived clones from the BM of NAA patients had very limited proliferative potential, while those of SAA patients did not differ from colonies of healthy donors. The analysis of CFU-F-derived colonies differentiation ability revealed that the proportion of the precursors that did not respond to the differentiation induction was higher in the BM of AA patients than in donors. It reflects the involvement of a certain subpopulation of stromal precursors that are either pre-differentiated into fibroblasts, or, conversely, earlier precursors of the hematopoietic microenvironment, which were not able to differentiate into osteoblasts and/or adipocytes within standard time. The analysis of the MMSC growth characteristics revealed that the time required for MMSC from SAA and NAA patients to form a confluent monolayer after the initial seeding and the population doubling time, were significantly higher than in MMSC of healthy donors. Thus, the proliferation rate of MMSC of AA patients is reduced. Nevertheless, the total cell production for 3 passages did not differ in cultures of AA patients and healthy donors. Therefore, the proliferative potential of MMSC of AA patients is not altered. Probably MMSC being analyzed ex vivo can restore their function. However, the analysis of REL of genes regulating the proliferation (FGF2, FGFR1, FGFR2) in MSCs had revealed the differences in comparison with donors and between SAA and NAA. Moreover, the analysis of the polymorphism in CFH gene, participating in immunomodulation, showed that the distribution differs between NAA and SAA patients. Conclusions. Stromal precursors in BM of untreated NAA and SAA patients are impaired and differ between the two subtypes of AA. It seems that the differences between NAA and SAA may lay not only in the absolute neutrophil count but also in the BM stroma itself. This effect could participate in the pathogenesis of AA or be the consequence of compensatory reaction of stromal microenvironment to the hematopoiesis failure. This work was supported by the Russian Foundation for Basic Research, project no. 19-015-00280. Table Disclosures No relevant conflicts of interest to declare.

Characteristics of Bone Marrow Progenitor Cells of C57BL/6N Mice after 30-Day Hindlimb Suspension and 12-Hour Readaptation to Support Loading

The functional status of bone marrow (BM) progenitor cells, which provide hematopoiesis and homeostasis of bone tissue, determines many processes which take a place in spaceflight (SF) and subsequent recovery to gravity. It is known that SF factors and simulation of their effects do irreversible damage to the pool of hematopoietic and stromal progenitor cells. Meanwhile, changes in the functional activity of BM progenitor cells after simulation of SF effects were more pronounced than in the case of a real SF. The aim of the study was to examine processes of recovery of the functional activity of BM progenitor cells during readaptaion to loading after 30-day hindlimb suspension. Studies were performed on three groups of male mice: vivarium control, suspension, and readaptation. The following characteristics were monitored: number of nucleated BM cells, immunophenotype, proliferative activity of stromal BM cells, number of hematopoietic colony-forming units (CFU), CFUs of fibroblasts, and differentiating capabilities of BM hematopoietic and stromal precursors. Hindlimb suspension resulted in decreased number of fibroblast and hematopoietic CFU, inhibition of spontaneous osteodifferentiation of stromal progenitors and decreased expression of stromal markers. The readaptaion period was accompanied with restoration of the functional activity of stromal progenitors, while complete recovery of hematopoiesis was not observed.

Reporting Guidelines, Review of Methodological Standards, and Challenges Toward Harmonization in Bone Marrow Adiposity Research. Report of the Methodologies Working Group of the International Bone Marrow Adiposity Society.

The interest in bone marrow adiposity (BMA) has increased over the last decade due to its association with, and potential role, in a range of diseases (osteoporosis, diabetes, anorexia, cancer) as well as treatments (corticosteroid, radiation, chemotherapy, thiazolidinediones). However, to advance the field of BMA research, standardization of methods is desirable to increase comparability of study outcomes and foster collaboration. Therefore, at the 2017 annual BMA meeting, the International Bone Marrow Adiposity Society (BMAS) founded a working group to evaluate methodologies in BMA research. All BMAS members could volunteer to participate. The working group members, who are all active preclinical or clinical BMA researchers, searched the literature for articles investigating BMA and discussed the results during personal and telephone conferences. According to the consensus opinion, both based on the review of the literature and on expert opinion, we describe existing methodologies and discuss the challenges and future directions for (1) histomorphometry of bone marrow adipocytes, (2) ex vivo BMA imaging, (3) in vivo BMA imaging, (4) cell isolation, culture, differentiation and in vitro modulation of primary bone marrow adipocytes and bone marrow stromal cell precursors, (5) lineage tracing and in vivo BMA modulation, and (6) BMA biobanking. We identify as accepted standards in BMA research: manual histomorphometry and osmium tetroxide 3D contrast-enhanced μCT for ex vivo quantification, specific MRI sequences (WFI and H-MRS) for in vivo studies, and RT-qPCR with a minimal four gene panel or lipid-based assays for in vitro quantification of bone marrow adipogenesis. Emerging techniques are described which may soon come to complement or substitute these gold standards. Known confounding factors and minimal reporting standards are presented, and their use is encouraged to facilitate comparison across studies. In conclusion, specific BMA methodologies have been developed. However, important challenges remain. In particular, we advocate for the harmonization of methodologies, the precise reporting of known confounding factors, and the identification of methods to modulate BMA independently from other tissues. Wider use of existing animal models with impaired BMA production (e.g., Pfrt−/−, KitW/W−v) and development of specific BMA deletion models would be highly desirable for this purpose.

Cytological and functional characteristics of fascia adipocytes in rats: A unique population of adipocytes

The dermal adipocytes, superficial fascia and subcutaneous adipose tissue (SAT) exist in the interspaces between the dermis and muscular fascia. They are adjacent to each other and traditionally recognized as one SAT. Recently, the dermal adipocyte was redefined as a unique population independent from the SAT. Also, we identified a novel type of adipogenic progenitors in rat superficial fascia. This study aimed to examine cytological and functional characteristics of fascial adipocytes in rats. Superficial fascia had no adipocytes in neonatal rats but gradually appeared numbers of adipocytes in growing rats. Adipogenic progenitors were found to reside in fascia and had strong ability in spontaneous and induced adipogenic differentiation in vitro. Differentiated fascial adipocytes versus subcutaneous or visceral adipocytes expressed increased adipose triglyceride lipase but decreased beta-adrenoreceptor, perilipin-1 and hormone-sensitive lipase (HSL), thus having high basal lipolysis but low lipolysis response to catecholamines. Phosphorylation of perilipin-1 and HSL and translocation of HSL to lipid droplets were attenuated in response to catecholamines rather than post-adrenoreceptoral lipolytic stimulators. The results suggested that superficial fascia was an origin of adipocytes with distinct developmental, cytological and functional characteristics. We proposed that fascial adipocytes could be considered as a unique population of adipocytes in the body. The fascia origin of adipocytes as an adipogenic model might logically explain fat neogenesis occurred at anatomical locations where originally exist no adipose tissues and thereby no adipose-derived stromal precursors. Also, the special histoanatomical relations and overlaps between the dermis, superficial fascia, SAT, and their adipocytes were discussed.