Human Bone Marrow Cells Research Articles

PS1234 APPLICATION OF FLOW SOM TO CLASSICAL FLOW CYTOMETRY: UNSUPERVISED ANALYSIS OF HEMATOPOIESIS AND HEMATOLOGICAL MALIGNANCIES

Background:Multiparameter flow cytometry (MFC) is an essential tool for the diagnosis and follow‐up of hematological malignancies. Numerous panels have been devised that best allow to achieve these goals. However, analysis of MFC graphs still heavily relies on the expertise of biologists and on specifically well adapted cascade‐gating strategies. Because of the development of mass cytometry, which generates data in a multidimensional environment of up to 50 dimensions, new strategies have been developed, that could be applied to classical MFC.Aims:In this study, the capacities of a rapid new tool for multidimensional analysis, FlowSOM (self‐organizing maps) have been combined to a classical MFC analysis tool, Kaluza®, for the identification of normal and diseased human bone marrow (BM) cell subpopulations.Methods:Normal BM samples collected during bone surgery were analyzed in MFC with four antibody panels adapted to the exploration of acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL) respectively. The listmode files obtained were merged and submitted to FlowSOM analysis, using R packages, which yielded trees of 100 nodes defining normal BM cell subsets. FlowSOM parameters were then transferred to Kaluza® for a bidimensional representation of the trees within which selected navigation could be performed with Kaluza® tools. In a second part of the study, the same stainings were performed on myelodysplastic or leukemic (AML or ALL) samples (diagnosis or follow‐up), which were submitted to FlowSOM together with the reference merged BM.Results:Node‐by‐node selection allowed for a precise definition of each of the unsupervised subsets generated by the software. This disclosed a selective segregation of delicate subsets such as non‐classical monocytes, hematogones or stem cells. Direct simultaneous comparison of diseased samples and normal BM allowed for a precise definition of abnormal cells (blasts) at diagnosis, and their immediate recognition as minimal residual disease in follow‐up samples.Summary/Conclusion:Flow SOM appears as a new and efficient tool for a rapid identification of BM cell subsets, highlighting differences from normal BM in dysplastic of leukemic samples. The node‐by‐node approach facilitates the definition of abnormal cells and their quantification, supervision intervening only after the generation of subsets delineated by artificial intelligence.

PS1235 RED BLOOD CELL GENERATION FROM HUMAN INDUCED PLURIPOTENT STEM CELLS

Background: Multiparameter flow cytometry (MFC) is an essential tool for the diagnosis and follow-up of hematological malignancies. Numerous panels have been devised that best allow to achieve these goals. However, analysis of MFC graphs still heavily relies on the expertise of biologists and on specifically well adapted cascade-gating strategies. Because of the development of mass cytometry, which generates data in a multidimensional environment of up to 50 dimensions, new strategies have been developed, that could be applied to classical MFC. Aims: In this study, the capacities of a rapid new tool for multidimensional analysis, FlowSOM (self-organizing maps) have been combined to a classical MFC analysis tool, Kaluza®, for the identification of normal and diseased human bone marrow (BM) cell subpopulations. Methods: Normal BM samples collected during bone surgery were analyzed in MFC with four antibody panels adapted to the exploration of acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL) respectively. The listmode files obtained were merged and submitted to FlowSOM analysis, using R packages, which yielded trees of 100 nodes defining normal BM cell subsets. FlowSOM parameters were then transferred to Kaluza® for a bidimensional representation of the trees within which selected navigation could be performed with Kaluza® tools. In a second part of the study, the same stainings were performed on myelodysplastic or leukemic (AML or ALL) samples (diagnosis or follow-up), which were submitted to FlowSOM together with the reference merged BM. Results: Node-by-node selection allowed for a precise definition of each of the unsupervised subsets generated by the software. This disclosed a selective segregation of delicate subsets such as non-classical monocytes, hematogones or stem cells. Direct simultaneous comparison of diseased samples and normal BM allowed for a precise definition of abnormal cells (blasts) at diagnosis, and their immediate recognition as minimal residual disease in follow-up samples. Summary/Conclusion: Flow SOM appears as a new and efficient tool for a rapid identification of BM cell subsets, highlighting differences from normal BM in dysplastic of leukemic samples. The node-by-node approach facilitates the definition of abnormal cells and their quantification, supervision intervening only after the generation of subsets delineated by artificial intelligence.

Thermoresponsive Stiffness Softening of Hierarchically Porous Nanohybrid Membranes Promotes Niches for Mesenchymal Stem Cell Differentiation.

Despite the attention given to the development of novel responsive implants for regenerative medicine applications, the lack of integration with the surrounding tissues and the mismatch with the dynamic mechanobiological nature of native soft tissues remain in the current products. Hierarchical porous membranes based on a poly (urea-urethane) (PUU) nanohybrid have been fabricated by thermally induced phase separation (TIPS) of the polymer solution at different temperatures. Thermoresponsive stiffness softening of the membranes through phase transition from the semicrystalline phase to rubber phase and reverse self-assembly of the quasi-random nanophase structure is characterized at body temperature near the melting point of the crystalline domains of soft segments. The effects of the porous structure and stiffness softening on proliferation and differentiation of human bone-marrow mesenchymal stem cells (hBM-MSCs) are investigated. The results of immunohistochemistry, histological, ELISA, and qPCR demonstrate that hBM-MSCs maintain their lineage commitment during stiffness relaxation; chondrogenic differentiation is favored on the soft and porous scaffold, while osteogenic differentiation is more prominent on the initial stiff one. Stiffness relaxation stimulates more osteogenic activity than chondrogenesis, the latter being more influenced by the synergetic coupling effect of softness and porosity.

Single-Molecule Real-Time (SMRT) Full-Length RNA-Sequencing Reveals Novel and Distinct mRNA Isoforms in Human Bone Marrow Cell Subpopulations.

Hematopoietic cells are continuously replenished from progenitor cells that reside in the bone marrow. To evaluate molecular changes during this process, we analyzed the transcriptomes of freshly harvested human bone marrow progenitor (lineage-negative) and differentiated (lineage-positive) cells by single-molecule real-time (SMRT) full-length RNA-sequencing. This analysis revealed a ~5-fold higher number of transcript isoforms than previously detected and showed a distinct composition of individual transcript isoforms characteristic for bone marrow subpopulations. A detailed analysis of messenger RNA (mRNA) isoforms transcribed from the ANXA1 and EEF1A1 loci confirmed their distinct composition. The expression of proteins predicted from the transcriptome analysis was evaluated by mass spectrometry and validated previously unknown protein isoforms predicted e.g., for EEF1A1. These protein isoforms distinguished the lineage negative cell population from the lineage positive cell population. Finally, transcript isoforms expressed from paralogous gene loci (e.g., CFD, GATA2, HLA-A, B, and C) also distinguished cell subpopulations but were only detectable by full-length RNA sequencing. Thus, qualitatively distinct transcript isoforms from individual genomic loci separate bone marrow cell subpopulations indicating complex transcriptional regulation and protein isoform generation during hematopoiesis.

Osteogenic Differentiation Potential of Human Bone Marrow and Amniotic Fluid-Derived Mesenchymal Stem Cells in Vitro & in Vivo.

BACKGROUND:Cell therapies offer a promising potential in promoting bone regeneration. Stem cell therapy presents attractive care modality in treating degenerative conditions or tissue injuries. The rationale behind this is both the expansion potential of stem cells into a large cell population size and its differentiation abilities into a wide variety of tissue types, when given the proper stimuli. A progenitor stem cell is a promising source of cell therapy in regenerative medicine and bone tissue engineering.AIM:This study aimed to compare the osteogenic differentiation and regenerative potentials of human mesenchymal stem cells derived from human bone marrow (hBM-MSCs) or amniotic fluid (hAF-MSCs), both in vitro and in vivo studies.SUBJECTS AND METHODS:Human MSCs, used in this study, were successfully isolated from two human sources; the bone marrow (BM) and amniotic fluid (AF) collected at the gestational ages of second or third trimesters.RESULTS:The stem cells derived from amniotic fluid seemed to be the most promising type of progenitor cells for clinical applications. In a pre-clinical experiment, attempting to explore the therapeutic application of MSCs in bone regeneration, Rat lumbar spines defects were surgically created and treated with undifferentiated and osteogenically differentiated MSCs, derived from BM and second trimester AF. Cells were loaded on gel-foam scaffolds, inserted and fixed in the area of the surgical defect. X-Ray radiography follows up, and histopathological analysis was done three-four months post- operation. The transplantation of AF-MSCs or BM-MSCs into induced bony defects showed promising results. The AF-MSCs are offering a better healing effect increasing the likelihood of achieving successful spinal fusion. Some bone changes were observed in rats transplanted with osteoblasts differentiated cells but not in rats transplanted with undifferentiated MSCs. Longer observational periods are required to evaluate a true bone formation. The findings of this study suggested that the different sources; hBM-MSCs or hAF-MSCs exhibited remarkably different signature regarding the cell morphology, proliferation capacity and osteogenic differentiation potentialCONCLUSIONS:AF-MSCs have a better performance in vivo bone healing than that of BM-MSCs. Hence, AF derived MSCs is highly recommended as an alternative source to BM-MSCs in bone regeneration and spine fusion surgeries. Moreover, the usage of gel-foam as a scaffold proved as an efficient cell carrier that showed bio-compatibility with cells, bio-degradability and osteoinductivity in vivo.

Genome editing in primary cells and in vivo using viral-derived Nanoblades loaded with Cas9-sgRNA ribonucleoproteins

Programmable nucleases have enabled rapid and accessible genome engineering in eukaryotic cells and living organisms. However, their delivery into target cells can be technically challenging when working with primary cells or in vivo. Here, we use engineered murine leukemia virus-like particles loaded with Cas9-sgRNA ribonucleoproteins (Nanoblades) to induce efficient genome-editing in cell lines and primary cells including human induced pluripotent stem cells, human hematopoietic stem cells and mouse bone-marrow cells. Transgene-free Nanoblades are also capable of in vivo genome-editing in mouse embryos and in the liver of injected mice. Nanoblades can be complexed with donor DNA for “all-in-one” homology-directed repair or programmed with modified Cas9 variants to mediate transcriptional up-regulation of target genes. Nanoblades preparation process is simple, relatively inexpensive and can be easily implemented in any laboratory equipped for cellular biology.

PRODUCTION AND ANALYSIS OF THE BIOLOGICAL PROPERTIES OF RECOMBINANT HUMAN GRANULOCYTE COLONY STIMULATING FACTOR CHIMERIC FORM

The aim of this work was to design and study biological properties of the recombinant human granulocyte colony stimulating factor (G-CSF), «linked» to apolipoprotein A-I (apoA-I) by a peptide linker, for obtaining in perspective a prolong form of the drug based on this cytokine. Material and methods . The nucleotide sequences of the genes encoding G-CSF and apoA-I were designed and optimized for expression in Pichia pastoris yeast using several computer programs. The assembly of the gene coding for the G-CSF-apoA-I chimeric cytokine, its cloning in the pPICZa-A vector, and expression in P. pastoris cells were performed using standard genetic engineering methods. Purification of the chimeric cytokine was carried out by two-stage ion-exchange chromatography. The biological activity of the chimera was determined in vitro on rat and human bone marrow cells (BMC) using flow cytometry, cell cycle analysis and myelograms. Results. A recombinant P pastoris X-33 yeast strain producing a chimeric cytokine containing the amino acid sequence G-CSF from the N-terminus, and mature human apoA-I from the C-terminus was constructed. In experiments on BMC of rat, it was shown that G-CSF-apoA-I increases the number of granulocytes in 1.8-2 times less compared with G-CSF. At the same time, the chimeric cytokine maintained the viability of monocytic and lymphocytic cells. Unlike G-CSF, the chimera increased the number of blast cells and normalized neutrophil segmentation, reducing the number of anomalies 1.5 times more efficiently. Conclusion . A new chimeric cytokine G-CSF-apoA-I was constructed, exhibiting the properties of not only a colony-stimulating factor, but also a growth factor, supporting the viability of other types of BMC.

Preparation and in vitro characterization of cross-linked collagen–gelatin hydrogel using EDC/NHS for corneal tissue engineering applications

Corneal disease is considered as the second leading cause of vision loss and keratoplasty is known as an effective treatment for it. However, the tissue engineered corneal substitutes are promising tools in experimental in vivo repair of cornea. Selecting appropriate cell sources and scaffolds are two important concerns in corneal tissue engineering. The object of this study was to investigate biocompatibility and physical properties of the bio-engineered cornea, fabricated from type-I collagen (COL) and gelatin (Gel). Two gelatin based hydrogels cross-linked with EDC/NHS were fabricated, and their physicochemical properties such as equilibrium water content, enzymatic degradation, mechanical properties, rheological, contact angle and optical properties as well as their ability to support human bone-marrow mesenchymal stem cells (hBM-MSCs) survival were characterized. The equilibrium water content and enzymatic degradation of these hydrogels can be easily controlled by adding COL. Our findings suggest that incorporation of COL-I increases optical properties, hydrophilicity, stiffness and Young's modulus. The viability of hBM-MSCs cultured in Gel and Gel: COL was assessed via CCK-8 assay. Also, the morphology of the hBM-MSCs on the top of Gel and Gel: COL hydrogels were characterized by phase-contrast microscopy. This biocompatible hydrogel may promise to be used as artificial corneal substitutes.

Long noncoding RNAs of single hematopoietic stem and progenitor cells in healthy and dysplastic human bone marrow.

Long noncoding RNAs (lncRNAs) are regulators of cell differentiation and development. The lncRNA transcriptome in human hematopoietic stem and progenitor cells is not comprehensively defined. We investigated lncRNAs in 979 human bone marrow-derived CD34+ cells by single cell RNA sequencing followed by de novo transcriptome reconstruction. We identified 3,173 lncRNAs in total, among which 2,365 were previously unknown, and we characterized lncRNA stem, differentiation, and maturation signatures. lncRNA expression exhibited high cell-to-cell variation, which was only apparent in single cell analysis. lncRNA expression followed a lineage-specific and highly dynamic pattern during early hematopoiesis. lncRNAs in hematopoietic cells closely correlated with protein-coding genes of known functions in the regulation of hematopoiesis and cell fate decisions, and the potential regulatory roles of lncRNAs in hematopoiesis were imputed by projection from protein-coding genes with a “guilt-by-association” approach. We characterized lncRNAs preferentially expressed in hematopoietic stem cells and in various downstream differentiated lineage progenitors. We also profiled lncRNA expression in single cells from patients with myelodysplastic syndromes and in aneuploid cells in particular. Our study provides a global view of lncRNAs in human hematopoietic stem and progenitor cells. We observed a highly ordered pattern of lncRNA expression and participation in regulation of early hematopoiesis, and coordinate aberrant messenger RNA and lncRNA transcriptomes in dysplastic hematopoiesis. (Registered at clinicaltrials.gov with identifiers: 00001620, 00001397)

Human bone marrow assessment by single-cell RNA sequencing, mass cytometry, and flow cytometry.

New techniques for single-cell analysis have led to insights into hematopoiesis and the immune system, but the ability of these techniques to cross-validate and reproducibly identify the biological variation in diverse human samples is currently unproven. We therefore performed a comprehensive assessment of human bone marrow cells using both single-cell RNA sequencing and multiparameter flow cytometry from 20 healthy adult human donors across a broad age range. These data characterize variation between healthy donors as well as age-associated changes in cell population frequencies. Direct comparison of techniques revealed discrepancy in the quantification of T lymphocyte and natural killer cell populations. Orthogonal validation of immunophenotyping using mass cytometry demonstrated a strong correlation with flow cytometry. Technical replicates using single-cell RNA sequencing matched robustly, while biological replicates showed variation. Given the increasing use of single-cell technologies in translational research, this resource serves as an important reference data set and highlights opportunities for further refinement.

Dual Targeting of Aurora Kinases with AMG 900 Exhibits Potent Preclinical Activity Against Acute Myeloid Leukemia with Distinct Post-Mitotic Outcomes.

Aurora kinase A and B have essential and non-overlapping roles in mitosis, with elevated expression in a subset of human cancers, including acute myeloid leukemia (AML). In this study, pan-aurora kinase inhibitor (AKI) AMG 900 distinguishes itself as an anti-leukemic agent that is more uniformly potent against a panel of AML cell lines than are isoform-selective AKIs and classic AML drugs. AMG 900 inhibited AML cell growth by inducing polyploidization and/or apoptosis. AMG 900 and aurora-B-selective inhibitor AZD1152-hQPA showed comparable cellular effects on AML lines that do not harbor a FLT3-ITD mutation. AMG 900 was active against P-glycoprotein-expressing AML cells resistant to AZD1152-hQPA and was effective at inducing expression of megakaryocyte-lineage markers (CD41, CD42) on human CHRF-288-11 cells and mouse Jak2 V617F cells. In MOLM-13 cells, inhibition of p-histone H3 by AMG 900 was associated with polyploidy, extra centrosomes, accumulation of p53 protein, apoptosis, and cleavage of Bcl-2 protein. Co-administration of cytarabine (Ara-C) with AMG 900 potentiated cell killing in a subset of AML lines, with evidence of attenuated polyploidization. AMG 900 inhibited the proliferation of primary human bone marrow cells in culture, with a better proliferation recovery profile relative to classic antimitotic drug docetaxel. In vivo, AMG 900 significantly reduced tumor burden in a systemic MOLM-13 xenograft model where we demonstrate the utility of 3'-deoxy-3'-18F-fluorothymidine [18F]FLT positron emission tomographic (PET)-CT imaging to measure the antiproliferative effects of AMG 900 in skeletal tissues in mice.

MRTFA Augments Megakaryocyte Maturation By Enhancing the SRF Regulatory Axis

Pediatric non-Down Syndrome acute megakaryoblastic leukemia (AMKL), a disease where megakaryocyte (MK) maturation is blocked, has a very poor prognosis. In 13% of AMKL cases of this type, the transcriptional co-factor MRTFA is expressed as part of a fusion protein. Normally, MRTFA levels increase in hematopoietic cells during megakaryopoiesis, making it likely that expression of MRTFA as part of a fusion protein leads to aberrant gene regulation, which results in leukemia. However, the mechanism by which megakaryopoiesis is blocked in AMKL is unknown. Therefore, we sought to parse out the role of MRTFA in normal megakaryopoiesis, so that we could better understand the cause for maturation block in leukemia.MRTFA is a co-factor of serum response factor (SRF). Knockout of either SRF or MRTFA in mice decreases MK maturation causing thrombocytopenia; and MRTFA overexpression (MRTFAOE) promotes MK maturation of primary human bone marrow (BM) cells in vitro. Our novel study shows that genomic regulation by MRTFA promotes MK maturation. In the human erythroleukemia (HEL) cell line, MRTFAOE enhances phorbol ester (TPA)-induced megakaryopoiesis, mimicking the effects of MRTFA on primary MK maturation. TPA-induced HEL cells with MRTFAOE achieve significantly higher 8N and 16N ploidy (p < 0.001, N = 4), compared to those without MRTFAOE. To identify the mechanisms underlying megakaryocytic differentiation, we bioinformatically analyzed anti-SRF chromatin immunoprecipitation (ChIP)-sequencing [approx. 15 million reads/sample] and RNA-sequencing data [approx. 20 million reads/sample], from non-induced and TPA-induced HEL cells, with and without MRTFAOE (N = 2/condition). We identified SRF peaks that change during TPA-induction, with and without MRTFAOE, and analyzed the strength of their binding and motif status. MRTFOE not only increased SRF binding to the genome during MK maturation (p ≤ 10-8), but preferentially retained binding at genomic CArG (CC[W]6GG) motifs, where SRF binds to in association with either MRTFA or specific ETS proteins (ELK1, ELK4). We then analyzed upregulated and downregulated genes during TPA-induction, with and without MRTFAOE, and identified those that had associated SRF peaks. As expected, TPA-induction upregulated megakaryocytic and cytoskeletal genes (VWF, ACTN1, CORO1A) and downregulated erythroid genes (KLF1, GYPB, GYPE). Interestingly, TPA-induction with MRTFAOE increased the number of upregulated genes by 27% and the number of downregulated genes by 10%. In TPA-induced cells, MRTFAOE increased the percentage of differentially expressed genes that had SRF peaks (25% versus 11% for upregulated genes, and 9% versus 4% for downregulated genes), further highlighting the direct role that MRTFA plays in MK maturation. Also, genes upregulated by TPA-induction alone have both ETS and CArG motifs, whereas those upregulated by TPA-induction with MRTFAOE lack ETS binding motifs, suggesting that MRTFAOE skews SRF binding toward CArG motifs. With anti-MRTFA ChIP-PCR in HEL cells, we confirmed the novel finding that along with SRF, MRTFA binds to regions associated with megakaryocytic and cytoskeletal genes (XRK6, CORO1A). Therefore, SRF and MRTFA together regulate expression of genes that are important for normal megakaryopoiesis, which explains why lack of these proteins adversely affects megakaryopoiesis in mice.We asked whether our findings in HEL cells were applicable to the more clinically relevant primary human BM cells. Anti-SRF and anti-MRTFA ChIP-PCR on day 0 primary human cells (CD34+ cells) and day 8 differentiated cell subpopulations (CD41+CD42-, CD41+CD42+) confirmed that both SRF and MRTFA have increased binding during megakaryopoiesis at target sites associated with upregulated genes, such as CORO1A, TNS1, and XRK6 (p < 0.001). Therefore, we illustrated that transcriptional regulation by SRF/MRTFA function similarly in human BM cells undergoing megakaryopoiesis.We show for the first time that MRTFA increases both the genomic association and activity of SRF, and upregulates genes that enhance primary human megakaryopoiesis. These findings suggest that aberrant expression of MRTFA as a fusion protein in AMKL may disrupt essential transcriptional regulation via the SRF/MRTFA axis, resulting in blocked MK maturation. This forms a crucial reference point for future studies to understand the altered SRF/MRTFA function in AMKL. DisclosuresNo relevant conflicts of interest to declare.

Specific Bone Marrow Mesenchymal Subsets in Patients with Myelodysplastic Syndromes Harbor Molecular Perturbations That Alter the Dynamics of Competition between Pre-Leukemic Clones and Normal Cells

Myelodysplastic syndromes (MDS) are clonal disorders of aberrant hematopoietic stem cells. The role of the hematopoietic microenvironment (HMEV) in the initiation and maintenance of MDS remains unclear, though model systems have provided strong evidence for the potential of specific mesenchymal subsets, mostly osteoprogenitor cells, within the HMEV as a driver of the disease. Distinct behavior of various models emphasizes the importance of analyzing specific and highly defined populations rather than bulk mesenchymal cells when investigating patient-derived bone marrow (BM) samples.We have developed a platform that allows us to prospectively isolate specific human BM mesenchymal subsets with a comparable transcriptional profile to mouse osteoprogenitor cells that have been shown to drive hematological malignancies when perturbed (Raaijmakers et al., 2010; Dong et al, 2016). Using a combination of three cell surface markers (CD271, CD146, CD106) that were previously shown to prospectively enrich for mesenchymal cells in human BM, we isolated and performed transcriptional and functional analysis of four distinct subsets within the non-hematopoietic (CD45, CD235), non-endothelial (CD31) compartment of the human BM. The four populations are labeled 1A (CD271+CD106-), 1B (CD271+CD106+), 2A (CD271+CD146+CD106-), and 2B (CD271+CD146+CD106+) (Figure 1A). Comparative analysis of differentially expressed gene sets based on RNA-seq within the human and mouse mesenchymal populations identified population 2B to be the most similar to osterix (OSX)-labeled mouse osteoprogenitor cells, whereas population 1A was more similar to osteocalcin (OCN)-labeled mouse mature osteoblasts. In animal models, genetic perturbations specifically in OSX but not OCN labeled cells resulted in hematological malignancy. Based on that observation, we hypothesized that population 2B is the cell subset most likely to harbor molecular perturbations in the BM of MDS patients. We analyzed populations 2A, 2B, 1(1A+1B) and another population 3 which is devoid of colony forming cells and encompasses all CD271-CD146- non hematopoietic stromal cells in 16 human MDS patients. BM from 11 patients undergoing hip replacement surgeries was used as a source of age- and gender-matched normal controls.Despite the heterogeneity of the disease, principal component analysis revealed that the transcriptional profile of normal samples clearly separated from MDS patients mostly in populations 2B and 1 (Figure 1B). Functional annotation of the differentially expressed genes using the Database for Annotation, Visualization and Integrated Discovery (DAVID) demonstrated minimal overlap of enriched terms between the four populations. Thus our data demonstrate transcriptionally distinct subsets of BM mesenchymal cells and identify signatures of transcriptional deregulation which differentiate these cell subsets in MDS patients from normal individuals.Osteopontin, an extracellular matrix protein expressed by osteoblasts among other cells, was the most significantly differentially expressed gene in population 2B of MDS patients.To study the effect of the gene expression perturbations on MDS evolution in vivo, we created a chimeric model where BM cells expressing the Nup98/HoxD13 (NHD13) translocation were transplanted in competition with wild type cells in wild type (WT) or osteopontin knock out (Opn-KO) recipients. Animals were conditioned using immunotoxin to avoid the damaging effect of irradiation on the microenvironment. Following chimerism of donor cells in peripheral blood over 24 weeks demonstrated a competitive advantage of NHD13 cells in the Opn-KO recipients leading to a significant difference in survival when compared to NHD13 cells transplanted in wild type mice (Figure 1C, D).Altogether, our data provide evidence that specific mesenchymal progenitor subsets in the BM of MDS patients harbor molecular perturbations that contribute to the competitive advantage of MDS over normal hematopoietic cells. DisclosuresSykes:Clear Creek Bio: Equity Ownership, Other: Co-founder.

Nocturnal Melatonin Renews Bone and Blood Forming Stem Cells Reservoir By Metabolic Reprograming

Bone marrow (BM) residing hematopoietic stem and progenitor cells (HSPC) replenish the blood with mature cells with a finite life span on a daily basis while maintaining the reservoir of undifferentiated stem cells. We recently showed that light/darkness onset induce two different BM HSPC peaks. Morning-induced norepinephrine and TNF secretion metabolically facilitate HSPC differentiation and egress to replenish the circulation with new mature leukocytes. Night augmented BM melatonin renews BM CD150+ hematopoietic stem cell (HSC) reservoir and their long-term repopulation potential (Golan et al, Cell Stem Cell, In Press). How melatonin primes BM HSPC to change their phenotype and function to re-acquire an undifferentiated and primitive state, is poorly understood. The hormone melatonin is an important mediator of bone formation and mineralization, and ultimately regulates the balance of bone remodeling (Cardinali DP et al, J. Pineal Res., 2003). The cross talk between HSPC and their BM stromal microenvironment is tightly regulated and determines HSPC fate. Therefore, we examined whether melatonin plays a role in regulation of murine BM mesenchymal stem and progenitor cells (MSPC, CD45-/Sca-1+/PDGFRα+), known to support HSPC maintenance in their BM niches. Mice treated with melatonin for 5h during the morning had increased levels of BM MSPC endowed with higher colony-forming unit fibroblast (CFU-F) potential in vitro. Interestingly, the metabolic state of these progenitor cells was altered by melatonin demonstrating reduced glucose uptake ability and lower mitochondria content. To test if differences in stromal cells content exist between day and night, we examined BM MSPC and found increased levels at 11PM, the time of melatonin BM peak, with higher Sca-1high surface expression levels, as compared to daylight 11AM. These changes were associated with augmented CFU-F levels by MSPC harvested at 11PM and accompanied by reduced glucose uptake levels and mitochondria content. Our preliminary results suggest that melatonin at night increases BM MSPC levels and reduces their metabolic activity to maintain them in a primitive and undifferentiated state. Moreover, we found that melatonin-elevated HSPC at 11PM also share lower glucose uptake ability with reduced mitochondria content and lower mitochondrial membrane potential (evaluated by TMRE). We hypothesize that melatonin reprograms the metabolic state of both HSPC and their stromal MSPC microenvironment to renew and maintain a primitive state of both populations at night. One of the factors inhibited by melatonin is the bioactive lipid Sphingosine 1-Phosphate (S1P), which in turn inhibits melatonin production. We found that mice with low S1P levels (S1Plow) due to lack of the SPHK1 enzyme have high BM melatonin levels also during the day in contrast to wild type (WT) mice. S1Plow mice had higher levels of primitive stromal progenitor cells including CFU-F and lower levels of differentiating osteoblast precursors compared to WT mice. In addition, these mice had less BM Reactive Oxygen Species (ROS)high committed hematopoietic progenitor cells, but more primitive ROSlow EPCR+ HSC endowed with higher long-term repopulation capacity in both primary and serially transplanted recipients. Next, we examined how light/dark cues affect the homing of transplanted BM HSPC into the BM of irradiated hosts 18h after transplantation. We found that donor HSPC harvested at 11PM have elevated homing ability compared to 11AM harvested cells. Importantly, MSPC also better homed to the BM of irradiated recipients when we transplanted donor BM cells obtained at 11PM compared with 11AM. As a result, accelerated BM repopulation kinetics was documented one week post transplantation in mice transplanted with BM cells harvested at 11 PM.Taken together, our results reveal that in vivo melatonin renews and maintains the BM reservoir and function of primitive MSPC and HSPC by metabolically reprogramming these cells during the night on a daily basis. Since the primed HSPC and MSPC at night showed improved function and BM homing potential, these features might be mimicked by human BM cells in order to harness them for improved clinical transplantation protocols. DisclosuresNo relevant conflicts of interest to declare.

Rapamycin Promote the Inhibition Rate of Proliferation on SHI-1 Cells and Enhance the Efficacy of Chemotherapy in Npg Leukemia Mice

Acute myeloid leukemia (AML) is a malignant hematologic disease. The remission rate of traditional chemotherapy is about 70-80%. In order to improve the efficacy of chemotherapy, some new drug which could enhance the efficacy of chemotherapy should be explored. Rapamycin is an immunosuppressive agent which can regulate cell proliferation, autophagy and other activities by inhibiting mTOR signal pathway. The studies of rapamycin as an anti-leukemia agent were limited. We had successfully establish a leukemia model in NPG mice with systemic leukemia infiltration using the human acute monocytic leukemia cell line SHI-1 cells. So in this study, the rapamycin and/or chemotherapy were used to treat the SHI-1 cells in vitro and NPG leukemia mice to investigate the efficacy of rapamycin in the treatment of AML. Rapamycin alone could effectively inhibit the proliferation of SHI-1 cells in vitro, and the inhibition rate were 35.4% which lower than the inhibition rate chemotherapy group (49.9%) using cytarabine and adriamycin. The combination with rapamycin and chemotherapy could significantly enhanced the inhibition rate to 60.3% (P<0.05) (Fig A). At day 19 after SHI-1 were inoculated to the NPG mice from tail vein, nearly 1% CD45 and CD33 positive cells were found in the peripheral blood. Then the NPG mice were divided to 4 groups to receive treatment. Control group without any treatment, rapamycin group were intraperitoneally injected with rapamycin (10mg/kg) every day for 5 days, chemotherapy group were intraperitoneally injected with cytarabine (100mg/kg for 5 days) and adriamycin (1mg/kg every other day for 3 times), combined group were treated with rapamycin, cytarabine and adriamycin. At the first day after treatment, one NPG mice were sacrificed randomly in four groups. The weight of the spleen in control group, rapamycin group, chemotherapy group and combination group were 78.6 mg, 48.9 mg, 16.1 mg, 11.7 mg respectively (Fig B). The ratio of human CD33 and CD45 positive cells in peripheral blood, bone marrow and spleen of NPG mice were 20.2%, 23.2% and 16.8% in control group, 2.02%, 2.68% and 3.31% in rapamycin group, 13.6%, 14.2% and 5.50% in chemotherapy group, 0.31%, 2.49% and 1.22% in combined group respectively (Fig C). Histopathological results showed that the degree of leukemia infiltration in the organs of NPG in the rapamycin group, chemotherapy group, and the combined group was significantly less than that of the control group. The median survival time of NPG mice in control group was 30.5 days. The treatment of rapamycin or chemotherapy could significantly prolong the media survival time of NPG mice to 35.0 and 34.0 days (P<0.05). When combined rapamycin with chemotherapy, the life time of NPG mice were significantly extended to 39 days (P<0.05) (Fig D). When NPG mice were died, more neoplasms were grow in the organs such as kidney, liver, stomach, mesenteric, mediastinum, bladder, spine, skin and neck in the NPG mice of control group, the treatment of rapamycin, chemotherapy and combined treatment would significantly decrease the counts of infiltrative organ with leukemia cells (Fig E). Altogether, our studies verified that rapamycin could inhibit the growth of SHI-1 cells and enhance the efficacy of chemotherapy in vitro. In NPG leukemia mice, the treatment of rapamycin alone could gain the efficacy as in chemotherapy group, when combined with chemotherapy, rapamycin had synergistic effect to significantly enhance the efficacy of chemotherapy to prolong the median survival time and decrease the degree of leukemia infiltration in NPG mice. DisclosuresNo relevant conflicts of interest to declare.

Proteome Analyses and Single-Cell RNA Sequencing Reveal Age-Dependent Re-Wiring of Central Carbon Metabolism in Myeloid-Biased Subsets of Human Hematopoietic Stem Cells

Background: Diminishing potential to replace damaged tissues is a hallmark for aging of somatic stem cells, but the mechanisms leading to aging remain elusive. Applying comprehensive proteomics studies on human hematopoietic stem and progenitor cells (HPCs) as well as 5 other cell populations that constitute the bone marrow niche, we have acquired a systems understanding of the mechanisms involved in aging of HPCs.Methods and materials: We present a proteome-wide atlas of age-associated alterations in HPCs along with 5 other cell populations including mesenchymal stromal cells (MSC) that comprise the bone marrow niche. For each, the abundance of a large fraction of the ~12,000 proteins identified was assessed in a cohort of 59 human subjects from different age groups. In selected samples, transcriptomics, metabolomics, and single cell RNA-Sequencing studies were simultaneously performed.Results:As the HPCs become older, one of the most prominent changes is the increase in abundance of proteins involved in the pathways for central carbon metabolism. This change is found only in HPCs and is reminiscent of the Warburg effect where glycolytic intermediates are rerouted towards pentose phosphate shunt and anabolism. Transcriptomic data confirm the increase in abundance of mRNA for the respective glycolytic enzymes in older HPCs. Metabolomics analyses provide further proofs demonstrating the trend towards anabolism upon aging. Altered abundance of early regulators of HPC differentiation reveals a reduced functionality and a bias towards myeloid differentiation at the expense of lymphoid development. Simultaneously, significant and complementary alterations in the bone marrow niche are observed. Whereas key factors responsible for homing, egress and adhesion of HPCs, e.g. SDF1/CXCL12, VCAM1, FN1, integrins α4, αL, β1 andβ2 decrease in abundance in MSCs, soluble factors responsible for myeloid differentiation, e.g.TGF-beta1, increase in abundance in the cellular niche with age. Transcriptomic analyses of single-sorted HPCs have demonstrated unequivocally that the mRNA levels of age-dependent increase in glycolytic enzymes are expressed at significantly higher levels in myeloid-biased versus than those in lymphoid-biased HPCs, whereas age-unaffected enzymes have similar mRNA levels in both subsets. The increase in abundance of glycolytic enzymes is hence linked with skewing of myeloid-biased HPCs in older human subjects.Conclusion: We have generated a comprehensive atlas of alterations in proteome landscapes of human HPCs and niche cells in bone marrow upon aging. In addition to findings that recapitulate the results derived from murine studies, the major novelties are (a) the alterations in central carbon metabolism in aging human HPCs, (b) the complementary decrease in levels of adhesive molecules and respective ligands in MSCs and HPCs. Single-cell studies have demonstrated that the age-related increase in abundance of the glycolytic enzymes is linked to the myeloid-biased HPCs. These data represent a valuable resource and serve as a basis for development of strategies targeting metabolic changes to enhance HPC regeneration. DisclosuresNo relevant conflicts of interest to declare.

Sphingosine-1-Phosphate Receptor 3 (S1PR3) Promotes Myeloid Commitment of Human Hematopoietic and Leukemic Stem Cells

Understanding the mechanisms underlying the abnormal differentiation of human acute myeloid leukemia (AML) may reveal novel therapies to eradicate leukemic stem cells (LSC), which are often resistant to standard treatments and contribute to relapse. Cellular metabolism is recognized as a hallmark of cancer and is known to be distinct between hematopoietic stem cells (HSC) and downstream progenitors. In particular, sphingosine-1-phosphate (S1P) is a bioactive lipid produced from sphingolipid metabolism that regulates proliferation and survival and is implicated in HSC egress, lymphocyte trafficking and mouse lymphoid lineage determination primarily through binding to S1PR1, one of five S1P G-protein coupled receptors. However, sphingolipids are understudied in human LSC and HSC. We recently found that sphingolipid perturbation governs HSC self-renewal and influences lineage outcome. Here, we show that S1PR3 governs myeloid commitment of LSC in a subset of human AML and is thus an attractive therapeutic target.Lipidomic analysis of LSC+ and LSC‒ fractions derived from AML patient samples and validated by xenotransplantation assays showed distinct sphingolipid alterations when compared to each other and to normal human cord blood (CB) CD34+CD38‒ stem cells and CD34+CD38+ progenitor populations. Interestingly, LSC+ fractions have increased S1P levels, prompting us to wonder if S1P signaling contributes to LSC maintenance. To gain a better understanding of the role of S1P in stemness, we examined S1P signaling in normal CB. Analysis of gene expression of S1PR1-5 and S1P transporters (SPNS2 and MFSD2B) within a comprehensive transcriptional roadmap of human hematopoiesis comprising thirteen normal populations from HSC to mature blood lineages demonstrated distinct expression patterns in specific blood lineages. S1PR1 and MFSD2B were most highly expressed in lymphoid and erythroid lineages, respectively, consistent with murine data. Notably, S1PR3 expression is specific to mature monocytes and granulocytes in human CB. S1PR3 protein was absent from the surface of long-term (LT) and short-term (ST)-HSC as determined by flow cytometry. Remarkably, lentiviral overexpression (OE) of S1PR3 was sufficient to promote myelopoiesis at the expense of erythropoiesis from LT- and ST-HSC in vitro in liquid culture, single cell assays and colony forming assays. To ascertain the mechanisms promoting myeloid commitment, we performed gene expression profiling by RNA-seq of LT- and ST-HSC following S1PR3 OE. This yielded a subset of shared genes similarly upregulated following S1PR3 OE relative to controls, including the known myeloid differentiation and AML-associated factors Early Growth Response 1 and 2 (EGR1/2) and Tribbles 1 (TRIB1). Thus, promiscuous expression of S1PR3 promotes a myeloid fate program in human HSC.S1PR3 protein expression was higher in AML patient samples relative to human CB and bone marrow cells by flow cytometry. Bioinformatic analysis of three independent AML cohorts revealed that AML patient samples with high S1PR3 gene expression also had high EGR2 and TRIB1 expression. Limiting dilution xenotransplantation assays of LSC-containing fractions sorted based on surface expression of S1PR3 demonstrated lower LSC frequency in S1PR3high versus S1PR3low/- LSC fractions. Moreover, S1PR3 OE in LSC+ fractions virtually abolished leukemic engraftment in xenotranplantation assays. These results suggest that higher S1PR3 levels in LSC are associated with a more mature myeloid state and that further increasing S1PR3 levels disrupts LSC maintenance. Treatment of mice bearing primary AML xenografts with FTY720, a S1P mimetic that targets S1P receptors including S1PR3, decreased leukemia burden in a subset of patient samples tested, including those obtained from relapsed and treatment-refractory patients. 70% of AML samples tested showed decreased LSC frequency in serial repopulation assays following FTY720 treatment. Importantly, treatment with FTY720 did not alter normal hematopoietic xenografts, demonstrating the existence of a therapeutic window. Collectively, our results provide the first direct evidence that sphingolipids govern myeloid commitment in human HSC and LSC, and demonstrate the potential of S1PR3 as a novel therapeutic target in AML for eradication of LSC while sparing HSC. DisclosuresNo relevant conflicts of interest to declare.

Neuroprotection by Human Dental Pulp Mesenchymal Stem Cells: From Billions to Nano.

Mesenchymal Stem Cell (MSC) therapy in recent years has gained significant attention. Though the functional outcomes following MSC therapy for neurodegenerative diseases are convincing, various mechanisms for the functional recovery are being debated. Nevertheless, recent studies convincingly demonstrated that recovery following MSC therapy could be reiterated with MSC secretome per se thereby shifting the dogma from cell therapy to cell "based" therapy. In addition to various functional proteins, stem cell secretome also includes extracellular membrane vesicles like exosomes. Exosomes which are of "Nano" size have attracted significant interest as they can pass through the bloodbrain barrier far easily than macro size cells or growth factors. Exosomes act as a cargo between cells to bring about significant alterations in target cells. As the importance of exosomes is getting unveil, it is imperial to carry out a comprehensive study to evaluate the neuroprotective potential of exosomes as compared to conventional co-culture or total condition medium treatments. Thus, the present study is designed to compare the neuroprotective potential of MSC derived exosomes with MSC-condition medium or neuron-MSC-co-culture system against kainic acid induced excitotoxicity in in vitro condition. The study also aims at comparing the neuroprotective efficacy of exosomes/condition medium/co-culture of two MSC viz., neural crest derived human Dental Pulp Stem Cells (hDPSC) and human Bone-Marrow Mesenchymal Stem Cells (hBM-MSC) to identify the appropriate MSC source for treating neurodegenerative diseases. Our results demonstrated that neuroprotective efficacy of MSC-exosomes is as efficient as MSC-condition medium or neuron-MSC co-culture system and treating degenerating hippocampal neurons with all three MSC based approaches could up-regulate host's endogenous growth factor expressions and prevent apoptosis by activating cell survival PI3K-B-cell lymphoma-2 (Bcl-2) pathway. Thus, the current study highlights the possibilities of treating neurodegenerative diseases with "Nano" size exosomes as opposed to transplanting billions of stem cells which inherit several disadvantages.

Establishment and characterization of immortalized erythroid progenitor cell lines derived from a common cell source

Immortalized erythroid progenitor cell lines, which exhibit potential for enucleated red blood cell (RBC) production, are expected to serve as an in vitro source of RBCs. These erythroid progenitor cell lines have previously been established from a variety of sources; however, large numbers of cell lines have not been established, characterized, and compared from a common cell source. In the present study, 37 cell lines were established from human bone marrow cells from a single donor. The time required for the establishment of each cell line varied greatly from 46 to 246 days. Of these lines, five were selected and their characteristics were analyzed. The cell lines established at the earliest time point showed better results in terms of both karyotype and differentiation potential than those established the latest. Moreover, obvious differences were noted even when cell lines were established at the earliest time point from the same source. These results suggest that it is important to select the best cell lines from ones established at the earliest time point for generating cell lines with low genomic abnormality and high differentiation ability. We have successfully generated an adult type of cell line with 50% cells carrying a normal karyotype and with 25% enucleation efficiency. These findings could be valuable in the development of an optimal method for establishing cell lines.

Calcium carbonate nanoparticles promote osteogenesis compared to adipogenesis in human bone-marrow mesenchymal stem cells

Rhombohedron-like and fusiform calcium carbonate nanoparticles were fabricated using a new method. Their geometry was controlled by varying the mixing speed and ratio of ethanol versus water in reaction system. The calcium carbonate nanoparticles (CCNPs) have slight effect on viability of human bone-marrow mesenchymal stem cells (hBMSCs) with dose-dependent and shape-dependent, but they can significantly promote osteogenic differentiation of hBMSCs in vitro by 10–37% increase of alkaline phosphatase (ALP) activity, 9–36% growth of collagen secretion and 1.13–1.83 folds upregulation of osteogenesis-related genes, even at lower dose ranges (5–20 μg/ml). The efficacity of promoting osteogenesis depends on the shape and dose of CCNPs. Furthermore, adipogenesis was inhibited by less accumulation of lipid droplets, lower triglyceride (TG) secretion and downregulation of adipogenesis-related genes. These findings improve the understanding of effects CCNPs on hBMSCs fate towards osteoblasts or adipocytes and have meaningful impact for combining use of CCNPs and hBMSCs in tissue engineering and regenerative medicine fields.