The Biology of COP Cells: Mesenchymal of Hematopoietic?

Circulating osteogenic precursor (COP) cells constitute a recently discovered population of circulating progenitor cells with the capacity to form not only bone but other mesenchymal tissues. A small but growing body of literature explores these cells, but with a great deal of disagreement and contradiction within it, mainly whether these cells are from mesenchymal or hematopoietic origin. This session will discuss the origins and biological characterization of these cells, including the identification strategies used to isolate these cells from the peripheral blood. It also examines the available knowledge on the in vitro and in vivo behaviour of these cells in plastic adherence, differentiation capacity, proliferation, and cellular homing. We will also review the profound and exciting implications for future use of COP cells in clinical practice, particularly in comparison with other types of stem cells.

Mechanism of miR-506 Targeting Autophagy and Apoptosis-Associated Gene Beclin1 to Promote Bone Marrow Mesenchymal Stem Cells (BMSCs) Differentiation and Metastasis to Breast Cancer

This study investigates miR-506 targeting the autophagy and apoptosis-related gene Beclin1 and analyzes the mechanism of its effect on bone marrow mesenchymal stem cells (BMSCs) differentiation and metastasis to breast cancer. Detection of miRNA-506 expression in BMSCs and breast cancer cells was done by Real-time PCR. A luciferase reporter system analyzed the targeting relationship between Beclin1 and miR-506. miR-NC group, BMSCs induction group, siRNA-NC group, and siRNA-Beclin1 group was set to measure Beclin1 expression, cell differentiation and migration by transwell assay, cell viability by MTT assay, proliferation by EdU staining and apoptosis and cycle by flow cell assay. miRNA-506 showed a high expression in breast cancer cells and low expression in BMSCs. miRNA-506 mimics significantly promote breast cancer cell proliferation which was inhibited by miRNA-506 inhibitors. The expression of Beclin1mRNA was significantly higher and miR-506 was lower in breast cancer cells. BMSCs induction significantly downregulated Beclin1 expression, increased miR-506 expression, and promoted cell invasive differentiation and metastatic capacity. In conclusion, elevated miR-506 expression was associated with decreased Beclin1 expression and increased metastatic differentiation capacity of breast cancer cells, which could effectively increase differentiation capacity and metastatic differentiation after induction by BMSCs.

DNMT3A R882 Mutation in Human Haematopoietic Stem Cells Alters Differentiation Towards Neutrophils and Monocytes

Age related clonal haematopoiesis (ARCH) is defined as the clonal expansion of hematopoietic stem cells (HSCs), driven by recurrent mutations. Many of these are also commonly seen in haematological malignancies, hence termed pre-leukemic mutations (pLM). ARCH carries an increased risk of haematological malignancies and cardiometabolic disease. Further understanding of the role of pLMs in HSC function is necessary to predict and possibly prevent leukemia. DNMT3A R882 is the most common pLM observed in ARCH and is associated with poor outcomes in acute myeloid leukemia (AML). It is acquired early in life and is positively selected in HSCs. HSCs of Dnmt3a knock-out or knock-in mouse models have increased self-renewal capacity and can differentiate towards all lineages. DNMT3A R882 mutation is observed in all mature blood lineages in ARCH individuals, indicating their multipotential differentiation capacity, but the dynamics of differentiation are not known. We hypothesise that DNMT3A R882 mutation affects the differentiation dynamics of HSCs, potentially contributing to disease risk. We characterised the functional changes in HSC differentiation driven by DNMT3A R882 at single cell resolution. Single phenotypic HSCs (CD33-/CD34+/CD45dim/CD38-/CD45RA-) from 9 individuals (Table 1) were cultured in media supporting differentiation into all major blood lineages. To assess changes in differentiation capacity between DNMT3A R882 and wild-type (WT) HSCs within each individual, each single-cell colony was genotyped by targeted DNA sequencing and scored by high-throughput flow cytometry. Flow cytometry data was analysed using a novel unbiased analytical pipeline, which we have named 'FlowPAC', allowing generation of a two-dimensional representation of the global output of HSC differentiation by identifying clusters based on fluorochrome intensity. Four samples were underpowered for either DNMT3A R882 or WT colonies and were excluded from this analysis. We did not observe any difference in erythroid versus myeloid or lymphoid (NK) differentiation capacity between DNMT3A R882 and WT HSC. DNMT3A R882 and WT HSCs also produced similar proportions of monocytic and neutrophil colonies. However, FlowPAC analysis identified differences in their level of maturity. CD15 (neutrophil marker) expression was significantly increased in myeloid clusters of DNMT3A R882 colonies compared to WT (p=0.0043), whereas CD14 (monocyte marker) expression was decreased compared to WT (p=0.0645). We further analysed mature neutrophil differentiation using CD66b expression. Consistent with promotion of neutrophil differentiation, CD66b median fluorescence intensity was significantly increased in the CD15+ population of DNMT3A R882 colonies compared to WT. To further investigate monocyte differentiation, we performed RNAseq on colonies containing only monocytes from 1 ARCH sample. Analysis of transcriptional profiles confirmed less mature monocytes in DNMT3A R882 colonies compared to WT. Overall, our data indicate that DNMT3A R882 mutation in HSCs alters neutrophil and monocyte production. Additionally, we observed that in contrast to samples with high LSC content leading to leukemic engraftment in mice, in samples where DNMT3A R882 HSCs were able to reconstitute normal blood production, the expression of the cell surface marker CD49f, a marker of the most potent HSCs, was significantly increased in DNMT3A R882 HSCs compared to WT at the time of sorting. This may suggest unequal distribution of this pLM within the preserved normal HSC pool prior to leukemic transformation. In conclusion, DNMT3A R882 alters the dynamics of human HSC myeloid differentiation. The propensity of DNMT3A R882 derived monocytes to retain an immature phenotype relative to WT cells could represent an early change later facilitating the complete differentiation block seen in AML, driven by the acquisition of additional mutations such as NPM1. DNMT3A R882 also promoted neutrophil maturation. Altered neutrophil differentiation has been observed in patients with germline DNMT3A mutation. As altered neutrophil function plays a key role in cardiovascular disease, future studies are required to investigate whether the effect of DNMT3A R882 on neutrophil differentiation may contribute to the increased cardiovascular disease risk associated with this mutation. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

Safe and Efficient Engraftment of CRISPR-Based ELANE Mono-Allelic Knocked out HSCs in Mice: Evidence for a Novel Treatment for ELANE Neutropenia

ELANE-related severe congenital neutropenia (SCN) is a disorder wherein numerous heterozygous mutations in the ELANE gene lead to misfolding and mislocalization of mutant neutrophil elastase, resulting in the death of myeloid cells and block in neutrophil differentiation. Currently, SCN is treated with daily injections of granulocyte colony-stimulating factor (G-CSF), but patients on this therapy are at risk of developing myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML). The only other effective therapy is allogeneic hematopoietic stem cell transplantation that may involve graft versus host disease and risk of serious infections. Thus, there is great interest in safe and efficient therapeutic alternatives. Emendo Biotherapeutics has developed a novel CRISPR-based (OMNI A1) ex-vivo gene editing strategy that involves specific excision of disease-causing ELANE mutant allele. This treatment was tested on CD34 + HSCs from SCN patients and resulted in a significant improvement in the maturation of myeloid cells and their differentiation to normal functional neutrophils. To assess the feasibility and safety of this editing strategy, we conducted an in vivo study using human HSCs derived from two healthy donors that were engrafted into a NOD scid gamma (NSG) immunocompromised mice model. The study involved a single iv injection of HSCs that were either not-treated (NT), electroporated without delivery of CRISPR nuclease (mock) or edited with Emendo's novel nuclease, OMNI A1,(excision). Each group consisted of 6 NSG female mice and an additional group of 4 mice served as vehicle control (no HSCs). All animals were subjected to total body irradiation, 1 day prior to the treatment, at a dose of 250 cGy. Animals were subjected to retro-orbital blood collection, at 4 designated time points during the study (Figure A), for CD45 + cell counts using FACS. All animals were evaluated for systemic reactions, clinical signs and body weight changes during the study period. At the end of the study period, animals were subjected to peripheral blood (PB), bone marrow (BM) and spleen sampling. FACS analysis of CD45 + cells was performed on those samples to determine the engraftment efficiency. Furthermore, excision levels for ELANE were measured in the engrafted cells by droplet digital PCR (ddPCR). Finally, multilineage differentiation capacity of the engrafted cells was evaluated by FACS. Analysis of human CD45 + cells, at 20 weeks, showed comparable levels of engraftment of the not-treated, mock treated and excised HSCs in the mouse PB, BM and spleen (No statistically significant difference, Kruskal Wallis test) (Figure B). Excised cells were detected in the mouse BM and excision levels in some of the mice were comparable to those measured in the HSCs prior to engraftment (about 30-36%). Human-derived cells from the BM and spleen of mice engrafted with excised HSCs showed similar multilineage differentiation capacity as obtained in mice engrafted with either NT or mock-treated HSCs. Engrafted human CD45 + cells differentiated into neutrophils (CD66b +), myeloid cells (CD33 +), Lymphocytes: B-cells (CD19 +) and T-cells (CD3 +). In addition, a population of engrafted hematopoietic CD34 + cells was detected in the BM. Excised HSCs of both donors gave rise to all lineages tested, as efficient as the NT and the mock groups. Finally, engrafted mice showed no systemic reactions, abnormal clinical signs or loss of body weight. These data show that edited cells can be engrafted successfully, maintain their excision profile and populate the bone marrow. This study also supports the safety of the OMNI A1 therapeutic composition. Figure 1 Figure 1. Disclosures Povodovski: Emendo Biotherapeutic: Current Employment. Makaryan: Emendo Biotherapeutic: Research Funding. Sabo: Emendo Biotherapeutic: Research Funding. Dicken: Emendo Biotherapeutic: Ended employment in the past 24 months. Herman: Emendo Biotherapeutic: Current Employment. Emmanuel: Emendo Biotherapeutic: Current Employment. Dale: X4 Pharmaceuticals: Consultancy, Honoraria, Research Funding.

Inflammatory Modulation of Polyethylene Glycol-AuNP for Regulation of the Neural Differentiation Capacity of Mesenchymal Stem Cells

A nanocomposite composed of polyethylene glycol (PEG) incorporated with various concentrations (~17.4, ~43.5, ~174 ppm) of gold nanoparticles (Au) was created to investigate its biocompatibility and biological performance in vitro and in vivo. First, surface topography and chemical composition was determined through UV-visible spectroscopy (UV-Vis), Fourier-transform infrared spectroscopy (FTIR), atomic force microscopy (AFM), scanning electron microscopy (SEM), free radical scavenging ability, and water contact angle measurement. Additionally, the diameters of the PEG-Au nanocomposites were also evaluated through dynamic light scattering (DLS) assay. According to the results, PEG containing 43.5 ppm of Au demonstrated superior biocompatibility and biological properties for mesenchymal stem cells (MSCs), as well as superior osteogenic differentiation, adipocyte differentiation, and, particularly, neuronal differentiation. Indeed, PEG-Au 43.5 ppm induced better cell adhesion, proliferation and migration in MSCs. The higher expression of the SDF-1α/CXCR4 axis may be associated with MMPs activation and may have also promoted the differentiation capacity of MSCs. Moreover, it also prevented MSCs from apoptosis and inhibited macrophage and platelet activation, as well as reactive oxygen species (ROS) generation. Furthermore, the anti-inflammatory, biocompatibility, and endothelialization capacity of PEG-Au was measured in a rat model. After implanting the nanocomposites into rats subcutaneously for 4 weeks, PEG-Au 43.5 ppm was able to enhance the anti-immune response through inhibiting CD86 expression (M1 polarization), while also reducing leukocyte infiltration (CD45). Moreover, PEG-Au 43.5 ppm facilitated CD31 expression and anti-fibrosis ability. Above all, the PEG-Au nanocomposite was evidenced to strengthen the differentiation of MSCs into various cells, including fat, vessel, and bone tissue and, particularly, nerve cells. This research has elucidated that PEG combined with the appropriate amount of Au nanoparticles could become a potential biomaterial able to cooperate with MSCs for tissue regeneration engineering.

Evaluation of the effect of donor weight on adipose stromal/stem cell characteristics by using weight-discordant monozygotic twin pairs

Background Adipose stromal/stem cells (ASCs) are promising candidates for future clinical applications. ASCs have regenerative capacity, low immunogenicity, and immunomodulatory ability. The success of future cell-based therapies depends on the appropriate selection of donors. Several factors, including age, sex, and body mass index (BMI), may influence ASC characteristics. Our aim was to investigate the effect of acquired weight on ASC characteristics under the same genetic background using ASCs derived from monozygotic (MZ) twin pairs. Methods ASCs were isolated from subcutaneous adipose tissue from five weight-discordant (WD, within-pair difference in BMI > 3 kg/m2) MZ twin pairs, with measured BMI and metabolic status. The ASC immunophenotype, proliferation and osteogenic and adipogenic differentiation capacity were studied. ASC immunogenicity, immunosuppression capacity and the expression of inflammation markers were investigated. ASC angiogenic potential was assessed in cocultures with endothelial cells. Results ASCs showed low immunogenicity, proliferation, and osteogenic differentiation capacity independent of weight among all donors. ASCs showed a mesenchymal stem cell-like immunophenotype; however, the expression of CD146 was significantly higher in leaner WD twins than in heavier cotwins. ASCs from heavier twins from WD pairs showed significantly greater adipogenic differentiation capacity and higher expression of TNF and lower angiogenic potential compared with their leaner cotwins. ASCs showed immunosuppressive capacity in direct cocultures; however, heavier WD twins showed stronger immunosuppressive capacity than leaner cotwins. Conclusions Our genetically matched data suggest that a higher weight of the donor may have some effect on ASC characteristics, especially on angiogenic and adipogenic potential, which should be considered when ASCs are used clinically.

Nanogold-Carried Graphene Oxide: Anti-Inflammation and Increased Differentiation Capacity of Mesenchymal Stem Cells

Graphene-based nanocomposites such as graphene oxide (GO) and nanoparticle-decorated graphene with demonstrated excellent physicochemical properties have worthwhile applications in biomedicine and bioengineering such as tissue engineering. In this study, we fabricated gold nanoparticle-decorated GO (GO-Au) nanocomposites and characterized their physicochemical properties using UV-Vis absorption spectra, FTIR spectra, contact angle analyses, and free radical scavenging potential. Moreover, we investigated the potent applications of GO-Au nanocomposites on directing mesenchymal stem cells (MSCs) for tissue regeneration. We compared the efficacy of as-prepared GO-derived nanocomposites including GO, GO-Au, and GO-Au (×2) on the biocompatibility of MSCs, immune cell identification, anti-inflammatory effects, differentiation capacity, as well as animal immune compatibility. Our results showed that Au-deposited GO nanocomposites, especially GO-Au (×2), significantly exhibited increased cell viability of MSCs, had good anti-oxidative ability, sponged the immune response toward monocyte-macrophage transition, as well as inhibited the activity of platelets. Moreover, we also validated the superior efficacy of Au-deposited GO nanocomposites on the enhancement of cell motility and various MSCs-derived cell types of differentiation including neuron cells, adipocytes, osteocytes, and endothelial cells. Additionally, the lower induction of fibrotic formation, reduced M1 macrophage polarization, and higher induction of M2 macrophage, as well as promotion of the endothelialization, were also found in the Au-deposited GO nanocomposites implanted animal model. These results suggest that the Au-deposited GO nanocomposites have excellent immune compatibility and anti-inflammatory effects in vivo and in vitro. Altogether, our findings indicate that Au-decorated GO nanocomposites, especially GO-Au (×2), can be a potent nanocarrier for tissue engineering and an effective clinical strategy for anti-inflammation.