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Human bone marrow (BM) plasma cells are heterogeneous, ranging from newly arrived antibody-secreting cells (ASCs) to long-lived plasma cells (LLPCs). We provide single-cell transcriptional resolution of 17,347 BM ASCs from five healthy adults. Fifteen clusters are identified ranging from newly minted ASCs (cluster 1) expressing MKI67 and high major histocompatibility complex (MHC) class II that progress to late clusters 5-8 through intermediate clusters 2-4. Additional ASC clusters include the following: immunoglobulin (Ig) M predominant (likely of extra-follicular origin), interferon responsive, and high mitochondrial activity. Late ASCs are distinguished by G2M checkpoints, mammalian target of rapamycin (mTOR) signaling, distinct metabolic pathways, CD38 expression, utilization of tumor necrosis factor (TNF)-receptor superfamily members, and two distinct maturation pathways involving TNF signaling through nuclear factor κB (NF-κB). This study provides a single-cell atlas and molecular roadmap of LLPC maturation trajectories essential in the BM microniche. Altogether, understanding BM ASC heterogeneity in health and disease enables development of new strategies to enhance protective ASCs and to deplete pathogenic ones.

HypothesisWe hypothesized that Lomecel-B, an allogeneic medicinal signaling cell (MSC) therapeutic candidate for Alzheimer's disease (AD), is safe and potentially disease-modifying via pleiotropic mechanisms of action.Key predictionsWe prospectively tested the predictions that Lomecel-B administration to mild AD patients is safe (primary endpoint) and would provide multiple exploratory indications of potential efficacy in clinical and biomarker domains (prespecified secondary/exploratory endpoints).Strategy and key resultsMild AD patient received a single infusion of low- or high-dose Lomecel-B, or placebo, in a double-blind, randomized, phase I trial. The primary safety endpoint was met. Fluid-based and imaging biomarkers indicated significant improvement in the Lomecel-B arms versus placebo. The low-dose Lomecel-B arm showed significant improvements versus placebo on neurocognitive and other assessments.InterpretationOur results support the safety of Lomecel-B for AD, suggest clinical potential, and provide mechanistic insights. This early-stage study provides important exploratory information for larger efficacy-powered clinical trials.

Abstract Background Native bone marrow (BM) mesenchymal stem/stromal cells (BM-MSCs) participate in generating and shaping the skeleton and BM throughout the lifespan. Moreover, BM-MSCs regulate hematopoiesis by contributing to the hematopoietic stem cell niche in providing critical cytokines, chemokines and extracellular matrix components. However, BM-MSCs contain a heterogeneous cell population that remains ill-defined. Although studies on the taxonomy of native BM-MSCs in mice have just started to emerge, the taxonomy of native human BM-MSCs remains unelucidated. Methods By using single-cell RNA sequencing (scRNA-seq), we aimed to define a proper taxonomy for native human BM non-hematopoietic subsets including endothelial cells (ECs) and mural cells (MCs) but with a focal point on MSCs. To this end, transcriptomic scRNA-seq data were generated from 5 distinct BM donors and were analyzed together with other transcriptomic data and with computational biology analyses at different levels to identify, characterize and classify distinct native cell subsets with relevant biomarkers. Results We could ascribe novel specific biomarkers to ECs, MCs and MSCs. Unlike ECs and MCs, MSCs exhibited an adipogenic transcriptomic pattern while co-expressing genes related to hematopoiesis support and multilineage commitment potential. Furthermore, by a comparative analysis of scRNA-seq of BM cells from humans and mice, we identified core genes conserved in both species. Notably, we identified MARCKS, CXCL12, PDGFRA, and LEPR together with adipogenic factors as archetypal biomarkers of native MSCs within BM. In addition, our data suggest some complex gene nodes regulating critical biological functions of native BM-MSCs together with a preferential commitment toward an adipocyte lineage. Conclusions Overall, our taxonomy for native BM non-hematopoietic compartment provides an explicit depiction of gene expression in human ECs, MCs and MSCs at single-cell resolution. This analysis helps enhance our understanding of the phenotype and the complexity of biological functions of native human BM-MSCs.

Adoptive transfer of cultured BMSCs was shown to be immune-suppressive in various inflammatory settings. Many factors play a role in the process, but no master regulator of BMSC-driven immunomodulation was identified. Consequently, an assay that might predict BMSC product efficacy is still unavailable. Below, we show that BMSC donor variability can be monitored by IL-10 production of monocytes/macrophages using THP-1 cells (immortalized monocytic leukemia cells) co-cultured with BMSCs. Using a mixed lymphocyte reaction (MLR) assay, we also compared the ability of the different donor BMSCs to suppress T-cell proliferation, another measure of their immune-suppressive ability. We found that the BMSCs from a donor that induced the most IL-10 production were also the most efficient in suppressing T-cell proliferation. Transcriptome studies showed that the most potent BMSC batch also had higher expression of several known key immunomodulatory molecules such as hepatocyte growth factor (HGF), PDL1, and numerous members of the PGE2 pathway, including PTGS1 and TLR4. Multiplex ELISA experiments revealed higher expression of HGF and IL6 by the most potent BMSC donor. Based on these findings, we propose that THP-1 cells may be used to assess BMSC immunosuppressive activity as a product characterization assay. [ABSTRACT FROM AUTHOR]

Background: Native bone marrow (BM) mesenchymal stem/stromal cells (BM-MSCs) participate in generating and shaping the skeleton and BM throughout the lifespan. Moreover, BM-MSCs regulate hematopoiesis by contributing to the hematopoietic stem cell niche in providing critical cytokines, chemokines and extracellular matrix components. However, BM-MSCs contain a heterogeneous cell population that remains ill-defined. Although studies on the taxonomy of native BM-MSCs in mice have just started to emerge, the taxonomy of native human BM-MSCs remains unelucidated. Methods: By using single-cell RNA sequencing (scRNA-seq), we aimed to define a proper taxonomy for native human BM non-hematopoietic subsets including endothelial cells (ECs) and mural cells (MCs) but with a focal point on MSCs. To this end, transcriptomic scRNA-seq data were generated from 5 distinct BM donors and were analyzed together with other transcriptomic data and with computational biology analyses at different levels to identify, characterize and classify distinct native cell subsets with relevant biomarkers. Results: We could ascribe novel specific biomarkers to ECs, MCs and MSCs. Unlike ECs and MCs, MSCs exhibited an adipogenic transcriptomic pattern while co-expressing genes related to hematopoiesis support and multilineage commitment potential. Furthermore, by a comparative analysis of scRNA-seq of BM cells from humans and mice, we identified core genes conserved in both species. Notably, we identified MARCKS, CXCL12, PDGFRA, and LEPR together with adipogenic factors as archetypal biomarkers of native MSCs within BM. In addition, our data suggest some complex gene nodes regulating critical biological functions of native BM-MSCs together with a preferential commitment toward an adipocyte lineage. Conclusions: Overall, our taxonomy for native BM non-hematopoietic compartment provides an explicit depiction of gene expression in human ECs, MCs and MSCs at single-cell resolution. This analysis helps enhance our understanding of the phenotype and the complexity of biological functions of native human BM-MSCs. [ABSTRACT FROM AUTHOR]

Bone marrow is a cell-rich tissue of the reticuloendothelial system essential in the homeostasis and accurate functioning of hematopoiesis and of the immune system; moreover, it is also rich in lipids because it contains marrow adipocytes. This work aimed to evaluate the detection of mycobacterial DNA in human bone marrow as a tool to understand the complex pathology caused by the main pathogen Mycobacterium tuberculosis (Mtb). Formalin-fixed paraffin-embedded human bone marrow samples were studied using both conventional PCR + hybridization and in situ PCR to figure out the cell distribution of the targeted DNA. Samples were retrospectively collected from HIV+ patients with microbiologically proved mycobacterial infection and from subjects without evidence of infection. Mycobacterium avium (Mav) as well as Mtb DNA was detected in both settings, including tissues with and without granulomas. We detected DNA from both mycobacterial species, using in situ PCR, inside bone marrow macrophages. Other cell types, including adipocytes, showed positive signals only for Mtb DNA. This result suggested, for the first time, that marrow adipocytes could constitute an ideal reservoir for the persistence of Mtb, allowing the bacilli to establish long-lasting latent infection within a suitable lipid environment. This fact might differentiate pathogenic behavior of non-specialized pathogens such as Mav from that of specialized pathogens such as Mtb. [ABSTRACT FROM AUTHOR]

Summary: Background: SARS-CoV-2 specific antibody-secreting plasma cells (PC) mediating specific humoral immunity have been identified in the human bone marrow (BM) after COVID-19 or vaccination against SARS-CoV-2. However, it remained unclear whether or not they acquire phenotypes of human memory plasma cells. Methods: SARS-CoV-2-specific human bone marrow plasma cells (BMPC) were characterised by tetramer-based, antigen-specific flow cytometry and FluoroSpot assay. Findings: SARS-CoV-2 spike-S1-specific PC were detectable in all tested BM samples of previously vaccinated individuals, representing 0.22% of total BMPC. The majority of SARS-CoV-2-specific BMPC expressed IgG and their specificity for the spike S1 protein indicated emergence from a systemic vaccination response. Of note, one-fifth of SARS-CoV-2-specific BMPC showed the phenotype of memory plasma cells, i.e., downregulated CD19 and present or absent CD45 expression. Interpretation: Our data indicate the establishment of phenotypically diverse SARS-CoV-2-specific PC in the human BM after basic mRNA immunization, including the formation of memory phenotypes. These results suggest the induction of durable humoral immunity after basic mRNA vaccination against SARS-CoV-2. Funding: The study was supported by funding by the DFG grants TRR130 TP24, ME 3644/8-1, and the Berlin Senate. SR received funding from DFG SFB-1444 C01 Central Service Project.

Hematopoiesis is regulated by the bone marrow (BM) stroma. However, cellular identities and functions of the different BM stromal elements in humans remain poorly defined. Based on single-cell RNA sequencing (scRNAseq), we systematically characterized the human non-hematopoietic BM stromal compartment and we investigated stromal cell regulation principles based on the RNA velocity analysis using scVelo and studied the interactions between the human BM stromal cells and hematopoietic cells based on ligand-receptor (LR) expression using CellPhoneDB. scRNAseq led to the identification of six transcriptionally and functionally distinct stromal cell populations. Stromal cell differentiation hierarchy was recapitulated based on RNA velocity analysis and in vitro proliferation capacities and differentiation potentials. Potential key factors that might govern the transition from stem and progenitor cells to fate-committed cells were identified. In situ localization analysis demonstrated that different stromal cells were localized in different niches in the bone marrow. In silico cell-cell communication analysis further predicted that different stromal cell types might regulate hematopoiesis through distinct mechanisms. These findings provide the basis for a comprehensive understanding of the cellular complexity of the human BM microenvironment and the intricate stroma-hematopoiesis crosstalk mechanisms, thus refining our current view on human hematopoietic niche organization.

Circulating, blood-borne SARS-CoV-2-reactive memory T cells in persons so far unexposed to SARS-CoV-2 or the vaccines have been described in 20-100% of the adult population. They are credited with determining the efficacy of the immune response in COVID-19. Here, we demonstrate the presence of preexisting memory CD4+ T cells reacting to peptides of the spike, membrane, or nucleocapsid proteins of SARS-CoV-2 in the bone marrow of all 17 persons investigated that had previously not been exposed to SARS-CoV-2 or one of the vaccines targeting it, with only 15 of these persons also having such cells detectable circulating in the blood. The preexisting SARS-CoV-2-reactive memory CD4+ T cells of the bone marrow are abundant and polyfunctional, with the phenotype of central memory T cells. They are tissue-resident, at least in those persons who do not have such cells in the blood, and about 30% of them express CD69. Bone marrow resident SARSCoV-2-reactive memory CD4+ memory T cells are also abundant in vaccinated persons analyzed 10-168 days after 1°-4° vaccination. Apart from securing the bone marrow, preexisting cross-reactive memory CD4+ T cells may play an important role in shaping the systemic immune response to SARS-CoV-2 and the vaccines, and contribute essentially to the rapid establishment of longlasting immunity provided by memory plasma cells, already upon primary infection. [ABSTRACT FROM AUTHOR]

Circulating, blood-borne SARS-CoV-2-reactive memory T cells in persons so far unexposed to SARS-CoV-2 or the vaccines have been described in 20-100% of the adult population. They are credited with determining the efficacy of the immune response in COVID-19. Here, we demonstrate the presence of preexisting memory CD4+ T cells reacting to peptides of the spike, membrane, or nucleocapsid proteins of SARS-CoV-2 in the bone marrow of all 17 persons investigated that had previously not been exposed to SARS-CoV-2 or one of the vaccines targeting it, with only 15 of these persons also having such cells detectable circulating in the blood. The preexisting SARS-CoV-2-reactive memory CD4+ T cells of the bone marrow are abundant and polyfunctional, with the phenotype of central memory T cells. They are tissue-resident, at least in those persons who do not have such cells in the blood, and about 30% of them express CD69. Bone marrow resident SARS-CoV-2-reactive memory CD4+ memory T cells are also abundant in vaccinated persons analyzed 10-168 days after 1°-4° vaccination. Apart from securing the bone marrow, preexisting cross-reactive memory CD4+ T cells may play an important role in shaping the systemic immune response to SARS-CoV-2 and the vaccines, and contribute essentially to the rapid establishment of long-lasting immunity provided by memory plasma cells, already upon primary infection.

Bone marrow is a cell-rich tissue of the reticuloendothelial system essential in the homeostasis and accurate functioning of hematopoiesis and of the immune system; moreover, it is also rich in lipids because it contains marrow adipocytes. This work aimed to evaluate the detection of mycobacterial DNA in human bone marrow as a tool to understand the complex pathology caused by the main pathogen Mycobacterium tuberculosis (Mtb). Formalin-fixed paraffin-embedded human bone marrow samples were studied using both conventional PCR + hybridization and in situ PCR to figure out the cell distribution of the targeted DNA. Samples were retrospectively collected from HIV+ patients with microbiologically proved mycobacterial infection and from subjects without evidence of infection. Mycobacterium avium (Mav) as well as Mtb DNA was detected in both settings, including tissues with and without granulomas. We detected DNA from both mycobacterial species, using in situ PCR, inside bone marrow macrophages. Other cell types, including adipocytes, showed positive signals only for Mtb DNA. This result suggested, for the first time, that marrow adipocytes could constitute an ideal reservoir for the persistence of Mtb, allowing the bacilli to establish long-lasting latent infection within a suitable lipid environment. This fact might differentiate pathogenic behavior of non-specialized pathogens such as Mav from that of specialized pathogens such as Mtb.

Abstract Microglia, the immune sentinel of the central nervous system (CNS), are generated from yolk sac erythromyeloid progenitors that populate the developing CNS. Interestingly, a specific type of bone marrow‐derived monocyte is able to express a yolk sac microglial signature and populate CNS in disease. Here we have examined human bone marrow (hBM) in an attempt to identify novel cell sources for generating microglia‐like cells to use in cell‐based therapies and in vitro modeling. We demonstrate that hBM stroma harbors a progenitor cell that we name stromal microglial progenitor (STR‐MP). STR‐MP single‐cell gene analysis revealed the expression of the consensus genetic microglial signature and microglial‐specific genes present in development and CNS pathologies. STR‐MPs can be expanded and generate microglia‐like cells in vitro, which we name stromal microglia (STR‐M). STR‐M cells show phagocytic ability, classically activate, and survive and phagocyte in human brain tissue. Thus, our results reveal that hBM harbors a source of microglia‐like precursors that can be used in patient‐centered fast derivative approaches.

The long-term impact of viruses residing in the human bone marrow (BM) remains unexplored. However, chronic inflammatory processes driven by single or multiple viruses could significantly alter hematopoiesis and immune function. We performed a systematic analysis of the DNAs of 38 viruses in the BM. We detected, by quantitative PCRs and next-generation sequencing, viral DNA in 88.9% of the samples, up to five viruses in one individual. Included were, among others, several herpesviruses, hepatitis B virus, Merkel cell polyomavirus and, unprecedentedly, human papillomavirus 31. Given the reactivation and/or oncogenic potential of these viruses, their repercussion on hematopoietic and malignant disorders calls for careful examination. Furthermore, the implications of persistent infections on the engraftment, regenerative capacity, and outcomes of bone marrow transplantation deserve in-depth evaluation.

The long-term impact of viruses residing in the human bone marrow (BM) remains unexplored. However, chronic inflammatory processes driven by single or multiple viruses could significantly alter hematopoiesis and immune function. We performed a systematic analysis of the DNAs of 38 viruses in the BM. We detected, by quantitative PCRs and next-generation sequencing, viral DNA in 88.9% of the samples, up to five viruses in one individual. Included were, among others, several herpesviruses, hepatitis B virus, Merkel cell polyomavirus and, unprecedentedly, human papillomavirus 31. Given the reactivation and/or oncogenic potential of these viruses, their repercussion on hematopoietic and malignant disorders calls for careful examination. Furthermore, the implications of persistent infections on the engraftment, regenerative capacity, and outcomes of bone marrow transplantation deserve in-depth evaluation. [ABSTRACT FROM AUTHOR]

The functions of bone marrow plasma cells (BMPC) beyond antibody production are not fully elucidated and distinct subsets of BMPC suggest potential different functions. Phenotypic differences were identified for human BMPC depending on CD19 expression. Since CD19 is a co-stimulatory molecule of the B-cell-receptor (BCR), and IgA+ and IgM+ BMPC express the BCR on their surface, we here studied whether CD19 expression affects cellular responses, such as BCR signaling and the expression of checkpoint molecules. We analyzed 132 BM samples from individuals undergoing routine total hip arthroplasty. We found that both CD19+ and CD19− BMPC expressed BCR signaling molecules. Notably, the BCR-associated kinase spleen tyrosine kinase (SYK) including pSYK was higher expressed in CD19+ BMPC compared to CD19− BMPC. BCR stimulation also resulted in increased kinase phosphorylation downstream of the BCR while expression of CD19 remained stable afterwards. Interestingly, the BCR response was restricted to IgA+ BMPC independently of CD19 expression. With regard to the expression of checkpoint molecules, CD19− BMPC expressed higher levels of co-inhibitory molecule programmed cell death protein-1 (PD-1) than CD19+ BMPC. IgA+ BMPC characteristically upregulated PD-1 upon BCR stimulation in contrast to other PC subsets and inhibition of the kinase SYK abrogated PD-1 upregulation. In contrast, expression of PD-1 ligand, B and T lymphocyte attenuator (BTLA) and CD28 did not change upon BCR activation of IgA+ BMPC. Here, we identify a distinct characteristic of IgA+ BMPC that is independent of the phenotypic heterogeneity of the subsets according to their CD19 expression. The data suggest that IgA+ BMPC underlie different regulatory principles and/or exert distinct regulatory functions.

The functions of bone marrow plasma cells (BMPC) beyond antibody production are not fully elucidated and distinct subsets of BMPC suggest potential different functions. Phenotypic differences were identified for human BMPC depending on CD19 expression. Since CD19 is a co-stimulatory molecule of the B-cell-receptor (BCR), and IgA+ and IgM+ BMPC express the BCR on their surface, we here studied whether CD19 expression affects cellular responses, such as BCR signaling and the expression of checkpoint molecules. We analyzed 132 BM samples from individuals undergoing routine total hip arthroplasty. We found that both CD19+ and CD19− BMPC expressed BCR signaling molecules. Notably, the BCR-associated kinase spleen tyrosine kinase (SYK) including pSYK was higher expressed in CD19+ BMPC compared to CD19− BMPC. BCR stimulation also resulted in increased kinase phosphorylation downstream of the BCR while expression of CD19 remained stable afterwards. Interestingly, the BCR response was restricted to IgA+ BMPC independently of CD19 expression. With regard to the expression of checkpoint molecules, CD19− BMPC expressed higher levels of co-inhibitory molecule programmed cell death protein-1 (PD-1) than CD19+ BMPC. IgA+ BMPC characteristically upregulated PD-1 upon BCR stimulation in contrast to other PC subsets and inhibition of the kinase SYK abrogated PD-1 upregulation. In contrast, expression of PD-1 ligand, B and T lymphocyte attenuator (BTLA) and CD28 did not change upon BCR activation of IgA+ BMPC. Here, we identify a distinct characteristic of IgA+ BMPC that is independent of the phenotypic heterogeneity of the subsets according to their CD19 expression. The data suggest that IgA+ BMPC underlie different regulatory principles and/or exert distinct regulatory functions. [ABSTRACT FROM AUTHOR]

There is considerable interest in the discovery of biomimetic materials that improve cell adhesion and reduce the time of bone integration of implants. In vitro bone cells biocompatibility was performed for the two types of biocomposites hydroxyapatite-titanium (HApTi) and hydroxyapatite-titanium with added calcium fructoborate (HApTiCaFb). In order to investigate the potential cytotoxicity of biomaterials, cell adhesion, proliferation, and osteogenic differentiation, human mesenchymal stem cells (MSCs) were used. Regarding the in vitro biocompatibility, a slight improvement in the phenotype was observed for the samples to which CaFb was added. [ABSTRACT FROM AUTHOR]

BACKGROUND: The dense microstructure of biological scaffolds and the limitation of cell growth microenvironment are the two major difficulties in the application of biological scaffolds in bone tissue repair. OBJECTIVE: To prepare fluffy hydroxyapatite/polylactic acid composite fiber scaffold, so that cells can easily enter into the scaffold and to realize three-dimensional culture of bone marrow mesenchymal stem cells. METHODS: Fluffy hydroxyapatite/polylactic acid composite scaffold was prepared by using modified electrospinning technology combined with biomineralization. The physical and chemical properties of the fiber scaffold were measured and observed. Human bone marrow mesenchymal stem cells were inoculated on the fluffy hydroxyapatite/polylactic acid composite scaffold and traditional hydroxyapatite/polylactic acid composite scaffold. Cell proliferation,adherence and morphological changes were detected. RESULTS AND CONCLUSION: (1) The thickness of hydroxyapatite coating in the fluffy hydroxyapatite/polylactic acid composite scaffold was about 8.3 μm, most of hydroxyapatite fibers were in discrete state with a diameter of 8-14 μm. The fibers were connected by pores, and the pore diameter was (65士35)μm. The surface area, porosity and water absorption of the scaffold were significantly higher than those of the traditional scaffold (P < 0.01). (2) After 12 hours of culture, the adherence of bone marrow mesenchymalstem cells on the two scaffolds was similar, 83% and 81% cells adhered on the traditional and fluffy scaffolds，respectively.(3) After 7 days of culture, the number of proliferated cells in the fluffy hydroxyapatite/polylactic acid composite scaffold was significantly more than that in the traditional hydroxyapatite/polylactic acid composite scaffold (P < 0.01). (4) After 7 days of culture, FDA staining and scanning electric microscopy showed that cell-cell independent shape appeared in the traditional scaffold. A large number of cells appeared in the fluffy scaffold and grew into cell clusters with high cell activity, which formed a cell-fiber construction. These results indicate that this new type hydroxyapalite/polylactic acid composite scaffold is beneficial for cell entry and proliferation. [ABSTRACT FROM AUTHOR]

Ischemic stroke is the leading cause of death and disability worldwide, with increasing incidence and mortality, imposing a significant social and economic burden on patients and their families. However, cerebral vascular occlusion leads to acute loss of neurons and destruction of synaptic structures. The limited treatment options cannot adequately address intra-neuronal mitochondrial dysfunction due to stroke. Therefore, stem cell-derived mitochondria transplantation plays an important role in neuronal protection and recovery after stroke, when combined with the intracranial and extracranial immunoregulatory effects of stem cell therapy, revealing the mechanism of transferred mitochondria in stem cells in protecting neurological function among chronic-phase ischemic stroke by affecting the endogenous apoptotic pathway of neuronal cells. This research elaborated on the mitochondrial dysfunction in neurons after ischemic stroke, followed by human bone marrow mesenchymal stem cells (hBMSC) rescued damaged neurons by mitochondrial transfer through tunneling nanotubes (TNTs), and the immunomodulatory effect of the preferential transfer of stem cells to the spleen when transplanted into the body.which created an immune environment for nerve repair, as well as improved neurological recovery after the chronic phase of stroke. This review is expected to provide a novel idea for applying intracranial stem cell transplantation in chronic-phase ischemic stroke treatment., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

Nestin-expressing stromal cells (NESCs) and Schwann cells in the bone marrow (BM) play crucial roles as a niche for normal hematopoietic stem cells in mice. It has been reported that both types of cells are decreased in myeloproliferative neoplasms in patients and also in a mouse model, whereas an increase in NESCs was reported in acute myeloid leukemia. It is thus of interest whether and how these BM stromal cells are structured in myelodysplastic syndromes (MDS). Here, we focused on NESCs and glial fibrillary acidic protein (GFAP)-expressing cells in the BM of MDS patients. We found a marked increase of NESCs in MDS with fibrosis (MDS-F) at a high frequency (9/19; 47.4%), but not in MDS without fibrosis (0/26; 0%). Intriguingly, in eight of the nine (88.9%) MDS-F cases with elevated NESCs, a majority of NESCs also expressed GFAP, with an additional increase in GFAP single-positive cells. Furthermore, in seven of them, we found a prominent structure characterized by neurofilament heavy chain staining surrounded by NESCs with GFAP expression. This structure may represent peripheral nerve axons surrounded by Schwann cells, and could be relevant to the pathophysiology of MDS-F. [ABSTRACT FROM AUTHOR]

During erythropoiesis, the molecular chaperone alpha-hemoglobin-stabilizing protein (AHSP) sequesters free alpha-hemoglobin (αHb) and prevents precipitation of excess αHb. While AHSP is linked to hereditary anemia, the pattern of expression during specific erythroblast stages is poorly understood. We investigated gene and protein expressions of AHSP throughout progressive maturation stages of erythroblasts in biphasic cultures of blood and bone marrow samples from healthy donors. Differentiating erythroblasts were periodically subjected to flow cytometry, Amnis imaging and RT-qPCR analyses. We made parallel in vivo validations from naive murine bone marrow cells. Percentages of AHSP+ erythroblasts, protein expressions and AHSP gene expressions are negligible on culture day 6 (CFU-Es) and progressively increases from culture days 8–12 (peaks on day 12) and declines on day 14. Notably, sub-cellular location of AHSP is both in the cytoplasm and nucleus in the early erythroblasts while in the late stages of maturation AHSP is found predominantly in the nucleus, being expelled with it during enucleation. As both human bone marrow and peripheral blood mononuclear cells (PBMC) derived erythroblasts demonstrated similar expression patterns, sampling of erythroblasts from day 11 cultures could portray erythroblast chronology and provide optimum representative stage specific expression patterns. PBMCs may be suitable for comparison studies of AHSP expression in pathologic erythropoiesi

We investigated the effects of conditioned medium (CM) from human bone marrow mesenchymal stem cells (h BMMSC)s and pulse wave photobiomodulation (PW PBM), applied alone or in combination, on the stereological parameters and gene expression of some growth factors, during wound healing in a streptozotocin (STZ)-induced rat model of type one diabetes mellitus (T1DM). T1DM was induced in 72 rats and two incisions were made in each animal. The rats were assigned to one of four groups: a control (placebo) group, a Laser group (890 nm, 80 Hz, 0.2 J/cm 2 ); a CM group, and a combined CM + Laser group. On post-surgical days 4, 7, and 15, skin samples were extracted for stereology and reverse transcription PCR (RT-PCR) analyses of gene expression of basic fibroblast growth factor (bFGF), hypoxia-inducible factor (HIF-1α), and stromal cell-derived factor-1α (SDF-1α). The stereological examinations of the proximal and distal wounds revealed significantly enhanced healing in all the treated groups, compared to the control group. The extent of healing was significantly greater in the CM + Laser group than in the other treatment groups. The RT-PCR results also indicated greater gene expression in the CM + Laser and Laser groups than in the CM and control groups. Application of CM and PW PBM, alone or in combination accelerated the process of wound healing in T1DM rats. The results of combined application of CM and PW PBM, indicated a synergistic effect, and the combination treatment was statistically more effective than single applications of CM or PW PBM. [ABSTRACT FROM AUTHOR]

The aim of this study was to apply a new instrumental approach to the analysis of human bone marrow for forensic purposes. A new screening method for the detection of more than 30 psychoactive drugs in bone marrow was developed and applied to case samples. The drugs used in this study belonged to the following groups: benzodiazepines (BZDs, n = 16), tricyclic antidepressants (TCAs, n = 5), selective serotonin reuptake inhibitors (SSRI, n = 4), serotonin-norepinephrine reuptake inhibitors (n = 1), anticonvulsants (n = 1), imidazopyridines (n = 1) and piperazines (n = 3). The sample preparation procedure involved microwave-assisted extraction (MAE) and the experimental settings were optimized using the simplex method. Separation and detection was carried out using a UHPLC-TOF system. The method were validated using marrow samples, and further applied in the analysis of three case samples, in which diazepam, nordiazepam, citalopram, doxepin and paroxetine were successfully detected. Finally the presented method is a good example of an assay that could potentially find an application in forensic analysis. [ABSTRACT FROM AUTHOR]

Thrombocytopenia Cargeeg is a rare autosomal dominant disorder and one of three thrombocytopenias caused by mutation of cytochrome c (Online Mendelian Inheritance in Man entry THC4). Our previous observations of platelet-like structures in the marrow space and early platelet production in vitro suggested that the low platelet phenotype in Thrombocytopenia Cargeeg subjects is caused by premature release of platelets into non-vascular regions of the bone marrow. We now show that two processes of platelet release occur in Thrombocytopenia Cargeeg subjects. Circulating platelets have a normal marginal microtubule coil, and cultured megakaryocytes derived from peripheral blood cells of Thrombocytopenia Cargeeg subjects form proplatelets normally and release platelets containing a marginal microtubule coil, consistent with effective platelet release via the proplatelet mechanism. In contrast, platelet-like structures within the extravascular bone marrow space have the dimensions of platelets but lack the marginal microtubule coil, suggesting abnormal proplatelet-independent platelet release. The mechanism of extravascular platelet release remains unclear. The failure to recapitulate this mechanism in vitro implies that the phenotype is not simply an intrinsic property of CYCS mutation-carrying megakaryocytes, but is dependent on the interaction between these cells and their environment. [ABSTRACT FROM AUTHOR]

Background: SARS-CoV-2 specific antibody-secreting plasma cells (PC) mediating specific humoral immunity have been identified in the human bone marrow (BM) after COVID-19 or vaccination against SARS-CoV-2. However, it remained unclear whether or not they acquire phenotypes of human memory plasma cells., Methods: SARS-CoV-2-specific human bone marrow plasma cells (BMPC) were characterised by tetramer-based, antigen-specific flow cytometry and FluoroSpot assay., Findings: SARS-CoV-2 spike-S1-specific PC were detectable in all tested BM samples of previously vaccinated individuals, representing 0.22% of total BMPC. The majority of SARS-CoV-2-specific BMPC expressed IgG and their specificity for the spike S1 protein indicated emergence from a systemic vaccination response. Of note, one-fifth of SARS-CoV-2-specific BMPC showed the phenotype of memory plasma cells, i.e., downregulated CD19 and present or absent CD45 expression., Interpretation: Our data indicate the establishment of phenotypically diverse SARS-CoV-2-specific PC in the human BM after basic mRNA immunization, including the formation of memory phenotypes. These results suggest the induction of durable humoral immunity after basic mRNA vaccination against SARS-CoV-2., Funding: The study was supported by funding by the DFG grants TRR130 TP24, ME 3644/8-1, and the Berlin Senate. SR received funding from DFGSFB-1444 C01 Central Service Project., Competing Interests: Declaration of interests HEM received public sector funding through the German Research Council (DFG) grants TRR130 TP24 and ME 3644/8-1; and through the Berlin Senate. SR received public sector funding through the German Federal Ministry of Education and Research (BMBF) grand SFB-1444 C01 Central Service Project–Cell and Tissue Harvesting: supply of functionally and immunologically characterised human samples for the CRC research projects. All other authors indicate that they have no competing interests., (Copyright © 2023 The Authors. Published by Elsevier B.V. All rights reserved.)

Background: The ability of mesenchymal stem cells (MSCs) to differentiate into many cell types, and modulate immune responses, makes them an attractive therapeutic tool for cell transplantation and tissue engineering.Objective: This project was designed for isolation, culture, and characterization of human marrow-derived MSCs based on the immunophenotypic markers and the differentiation potential.Methods: Bone marrow of healthy donors was aspirated from the iliac crest. Mononuclear cells were layered over the Ficoll-Paque density-gradient and plated in tissue cultures dish. The adherent cells expanded rapidly and maintained with periodic passages until a relatively homogeneous population was established. The identification of adherent cells and the immune-surface markers was performed by flow cytometric analysis at the third passage. The in vitro differentiation of MSCs into osteoblast and adipocytes was also achieved.Results: The MSCs were CD11b (CR3), CD45, CD34, CD31 (PCAM-1), CD40, CD80 (B7-1), and HLA-class II negative because antigen expression was less than 5%, while they showed a high expression of CD90, and CD73. The differentiation of osteoblasts, is determined by deposition of a mineralized extracellular matrix in the culture plates that can be detected with Alizarin Red. Adipocytes were easily identified by their morphology and staining with Oil Red.Conclusion: MSCs can be isolated and expanded from most healthy donors, providing for a source of cell-based therapy.