Primary Mesenchymal Stromal Cells Research Articles

Abstract 1048: Targeting BCL-2 and CD38 in models of acute myeloid leukemia reduces tumour burden

Abstract The prognosis for patients with acute myeloid leukemia (AML) remains poor with high mortality rates. It is often not possible to achieve complete remission with current therapy and relapse following treatment is common. New and more targeted treatment approaches are therefore needed to improve outcomes for patients. AML progression and treatment resistance has been associated with overexpression of BCL2. Venetoclax, a BH3 mimetic targeting BCL2, has been shown to effectively target AML cells and induce cell death. It has been approved for the treatment of AML but as with other AML treatments not all patients respond, and others develop treatment resistance. Research has therefore focussed on exploring combination therapies for Venetoclax. Our group has previously shown that mitochondrial transfer from mesenchymal stromal cells (MSC) to AML blasts promotes AML growth and is mediated by CD38. Daratumumab targets CD38 and inhibits this transfer, which results in impaired AML growth and improved animal survival. Here, we investigate the effect of inhibiting both CD38 with daratumumab and BCL2 with Venetoclax on the survival of AML. Primary AML blasts and MSC were isolated from patients' bone marrow in accordance with the Declaration of Helsinki. Flow cytometry was used to measure CD38 and BCL2 expression in AML blasts compared to normal CD34+ progenitor cells. BCL2 expression was significantly higher in AML blasts and CD38 expression was also increased. Cell viability was significantly reduced following treatment with Venetoclax alone, whilst Daratumumab alone or in combination with Venetoclax did not affect AML survival further. However, the action of daratumumab relies on the bone marrow microenvironment and AML blasts were therefore co-cultured with MSC and then treated with Venetoclax or daratumumab or in combination. After 24 hours cells were stained with Annexin V-FITC/PI and flow cytometry was used to assess levels of apoptosis. Combination treatment with Venetoclax and Daratumumab resulted in significantly more apoptosis in AML cells compared to AML cells treated with single agent. Finally, the effect of combination treatment with Venetoclax and Daratumumab was assessed in vivo using an NSG xenograft mouse model of AML. Mice were engrafted with MV411-luc or patient derived AML and then treated with vehicle control (PBS) or daratumumab alone (5mg/kg on day 7 and 14) or Venetoclax alone (100mg/kg/day) or both daratumumab and Venetoclax. Bioluminescence imaging was used to assess disease engraftment and progression before and after treatment. Combining treatment with Daratumumab and Venetoclax in vivo significantly reduced tumour burden and improved animal survival compared to control or single agent. This data supports that combination treatment with Venetoclax and Daratumumab could have an important clinical application in the treatment of AML. Citation Format: Charlotte Hellmich, Jayna J. Mistry, Amelia Lambert, Jamie A. Moore, Aisha Jibril, Angela Collins, Kristian M. Bowles, Stuart A. Rushworth. Targeting BCL-2 and CD38 in models of acute myeloid leukemia reduces tumour burden [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1048.

Azacitidine Directly Acts on the Mesenchymal Stromal Cell Compartment in Myelodysplastic Syndromes to Alter Niche Function and Suppress Malignant Hematopoeitic Stem/Progenitor Cells

Background: Hematopoietic stem/progenitor cells (HSPC) are highly dependent on interaction with the specific bone marrow microenvironment (niche) which supports their survival and directs their differentiation. Increasing evidence suggests that the niche plays a central role in the development of clonal stem cell disorders such as myelodysplastic syndromes (MDS). Malignant clonal HSPC in MDS have been shown to reprogram mesenchymal stromal cells (MSC) to promote disease progression. In turn, the altered stromal microenvironment in MDS may directly impair healthy hematopoiesis. Accordingly, MSC isolated from MDS patients have shown phenotypical and functional abnormalities, which were not observed in patients successfully treated with azacitidine (AZA), currently the standard of care for higher-risk MDS patients. Although AZA is a potent inhibitor of DNA methyltransferases and is thus considered a hypomethylating agent, the exact anti-leukemic mechanisms are not understood and a clear correlation between DNA demethylation and response to AZA has not been demonstrated thus far. Hence, we looked beyond the current focus of AZA effects on clonal hematopoiesis and hypothesized that AZA may also act through direct modulation of the MSC compartment. To answer this question, we examined primary MSC treated in vitro with AZA as well as the functional interaction between MSC and HSPC after AZA treatment.Methods: Primary MSC were isolated from bone marrow of untreated MDS patients or age-matched healthy controls, expanded by plastic adherence, cultured until confluency under defined conditions and used for up to 4 passages. Identity of MSC was confirmed by presence of characteristic cell surface markers CD73, CD271, CD105 and CD90 and absence of CD45 and CD34 by flow cytometry. MSC were differentiated into the adipogenic or osteogenic lineage after in vitro treatment with AZA (10 uM). Co-culture experiments with CD34+ HSPC from MDS patients or healthy controls were performed on AZA-treated or untreated MDS-MSC or healthy MSC, respectively. After four days, CD34+ cells were harvested and assessed by colony-forming unit assays in methylcellulose. Single colonies were genotyped for MDS-associated mutations known to be present in the bone marrow sample. Each colony was genotyped for one or two known MDS-associated mutations and an input patient specimen was used as a control. Finally, RNA sequencing was performed on a mesenchymal stromal cell line (EL08-1D2), either untreated or treated with AZA for 4 days in vitro to uncover pathways affected by AZA in stromal cells.Results: MDS-MSC showed a decreased proliferative capacity as well as markedly impaired differentiation into the osteoblastic lineage compared to healthy MSC. Treatment with AZA had no effect on cell surface marker expression or viability of either healthy or MDS-MSC. AZA treatment significantly reduced the ability of MDS-MSC to differentiate towards the adipogenic lineage while osteogenic differentiation was severely impaired with or without AZA treatment. In contrast, osteogenic differentiation of healthy MSC was enhanced after AZA treatment. In co-culture experiments pretreatment with AZA significantly improved stromal support of healthy CD34+ HSPC while malignant MDS-HSPC were suppressed. RNA sequencing analysis revealed differential gene expression in AZA-treated vs untreated stroma with significantly more genes upregulated in AZA-treated samples. Specific changes in pathways involved in ECM-receptor interaction, cell adhesion molecules (CAMs), cytokine-cytokine receptor interaction and metabolic pathways in AZA treated EL08-1D2 stromal cells were determined and validated in primary MDS MSC samples.Conclusion: We show here that AZA directly modulates altered MSC function in MDS, thus influencing interaction with HSPC and leading to suppression of malignant hematopoiesis. Our data suggest an additional mode of action by which AZA exerts its therapeutic activity. To our knowledge this analysis is the first to examine the direct effect of AZA treatment on MSC. DisclosuresGötze:BMS: Honoraria; Celgene: Honoraria; Novartis: Honoraria; Amgen: Honoraria; Abbvie: Honoraria.

Adipocyte Induced Antagonism of ALL Growth Highlights a Novel Inhibitory Niche and Potential Therapeutic Opportunity

The importance of Bone Marrow Microenvironment (BMM) in driving ALL progression is widely recognised however the nature of the leukaemia niche remains unclear. To gain specific insight, we assessed the evolving composition of ALL- BMMs both in primary human disease and experimental models. Strikingly, BMMs in both human ALL (n=6) and Nalm-6 ALL xenografts (n =5) exhibited severely depleted adipocyte densities (98.2 and 75.7% reduction vs control, p <0.05) with adipocyte homeostasis resetting upon remission attainment. To assess the potential biological significance of the inverse correlation between ALL and adipocytes we co-cultured a panel of ALL cell lines (Nalm-6, REH and RS4;11) with both murine 3T3-L1 adipocytes as well as primary adipocytes differentiated from normal BM derived Mesenchymal stromal cells (MSC), assessing cellular outcomes. Surprisingly, both 3T3-L1 and BM-MSC derived adipocytes impaired growth of ALL cell lines in a time dependant fashion. In contrast, co-culture with primary osteoblasts and MSCs did not alter basal rates of cellular growth confirmingthat adipocytes inhibited ALL growth in a tissue specific manner.Mechanistically, adipocytes repressed the G1/G2/S/M cell cycle progression of ALL cells without enhancing apoptosis indicating that cell extrinsic signals derived from adipocytes induce cytostasis. Thus, impairment of adipocyte BMM's during ALL development may function to disable a stromally derived ALL suppressor. To further investigate the leukaemia inhibitory function of tissue adipocytes, we compared leukaemia development in adipocyte rich (tail BM, omental and gonadal fat) vs adipocyte poor environments (femoral/thoracic bones) using a xenotransplantation model (Figure 1). In vivo, Nalm-6 growth was significantly restrained in adipocyte rich tissue compared to adipocyte poor regions and adipocyte poor extramedullary control tissue. Similarly, patient derived xenografts (n =3) demonstrated delayed development in adipocyte rich regions recapitulating the cellular response of ALL cell lines. Consistent with the in vitro data, adipocyte imprinted ALL cells in vivo resided in a less active cell cycle state supporting the contention that adipocytes are a transducer of negative growth signals. To investigate the tumour suppressor mechanism controlling growth we performed transwell experiments revealing that, basal rates of cellular proliferation and cell cycling, could be restored following transwell separation consistent with contact/close proximity factor mediating adipocyte induced tumour suppressive signalling. In line with these results, RNA-Seq demonstrated upregulation in extracellular matrix receptor pathways (FDR q-val 0.14) together with downregulation of cell cycle genes (p <0.05) in adipocyte exposed ALL cell lines. Adipocytes correspondingly were reprogrammed by ALL environments as assessed by quantitative proteomics indicating a cooperative interplay between these cells. To further delineate the mechanism by which adipocytes are depleted in ALL-BMMs we investigated the MSC axis hypothesising that adipocyte development was disrupted. Significantly fewer MSCs expanded in ALL BM vs age matched control with a corresponding reduction in CFU's indicating that ALL deregulates the intrinsic self-renewal capacity of MSCs. We further compared the multidifferentiation potential of ALL-MSC vs control MSCs following developmental induction. Surprisingly we found that ALL-MSCs had enhanced capacity for adipogenic differentiation (3.93 ± 0.69 fold increase; n = 5; p= 0.01) without reciprocal disturbance in osteogenesis. Increased adipocyte commitment could be reproduced by exposing normal MSCs to leukaemic serum but not HSC cultures suggesting that the altered priming of ALL- MSCs towards adipocyte differentiation is induced via a leukaemia soluble factor. Collectively, our data suggests adipocyte depletion in leukaemia BMMs may be contributed to by repressed MSC growth and that whilst adipocyte primed MSC's accumulate, differentiation might be stalled through a blast specific mechanism.ConclusionOur results suggest that adipocyte microenvironments create an inhibitory niche for ALL cells consistent with a tumour suppressive function, dissecting how these inhibitory niches are disabled to promote ALL development could uncover a novel therapeutic avenue. [Display omitted] DisclosuresGribben:Karyopharm: Honoraria; Genentech/Roche: Honoraria; Abbvie: Honoraria; Acerta: Honoraria; Janssen: Honoraria; Kite: Honoraria; Pharmacyclics: Honoraria; TG Therapeutics: Honoraria; Celgene: Honoraria.

Soluble CX3CL1/Fractalkine Is a New Player in the Pro-Inflammatory Microenvironment of Multiple Myeloma Patients Related to Bone Marrow Plasma Cell Infiltration and Neo-Vascularization

Multiple myeloma (MM) is characterized by the tight dependence of plasma cells (PCs) to the bone marrow (BM) microenvironment that activates the production of several pro-inflammatory and growth factors involved in MM cell survival, bone remodeling alterations and increased angiogenesis. CX3CL1/Fractalkine is a unique chemokine synthetized as a membrane-bound protein and released as soluble protein after proteolytic cleavage by two metalloproteases as ADAM10 and ADAM17. CX3CL1 interacts with its single receptor CX3CR1 being involved in several physiological and pathophysiological processes including inflammation and angiogenesis. The role CX3CL1/CX3CR1 axis in MM is still unexplored and unknown.In this study, firstly we analyzed BM plasma levels of CX3CL1 in MM patients as compared to indolent monoclonal gammopathies and healthy controls. BM plasma was obtained from 38 MM patients (median age 70 years; 53% female and 47% male; ISS I= 32%, II= 34%, III=34%) 25 of them with newly diagnosed and 13 relapsed MM, and from sex-aged matched 14 Smoldering MM (SMM) patients and 11 monoclonal gammopathy of undetermined significance (MGUS) patients. 10 healthy donors (HD) (median age 70 years; 20% female and 80% male) were used as controls. We found that plasma levels were significantly increased in the BM plasma from MM patients compared to HD (median levels 0.88 ng/ml vs 0.56 ng/ml, p=0.004). Furthermore, increased CX3CL1 levels were observed in patients with active MM as compared to patients with asymptomatic disease, as MGUS and SMM (median levels MM vs MGUS: 0.88 ng/ml vs 0.67 ng/ml, p=0.0176; median levels MM vs SMM: 0.88 ng/ml vs 0.65ng/ml, p= 0.0329). Moreover, CX3CL1 levels increased in the advanced stages of disease (median levels ISS III vs ISS I: 1.18 ng/ml vs 0.72 ng/ml, p=0.0115; median levels ISS III vs ISS II: 1.18 ng/ml vs 0.74 ng/ml, p=0.0157). Accordingly, BM soluble CX3CL1 levels were positively correlated with the percentage of BM plasma cells in the cohort of MM patients (p=0.002). Subsequently, the possible link between soluble CX3CL1/fractalkine and BM angiogenesis has been investigated. CD34 expression was evaluated by immunohistochemistry on bone biopsies obtained from 28 MM patients, showed that BM plasma levels of CX3CL1 significantly correlated with both the number of CD34 positive vessels (p= 0.002) and the micro-vessels density (p=0.002). Thereafter, we performed experiments on chorioallantoic membranes to demonstrate the role of CX3CL1 in MM-induced angiogenesis. The incubation of BM plasma of MM patients for 12 days resulted in a significant angiogenic response in the form of numerous allantoic neovessels that were significantly inhibited by blocking anti-CX3CL1 antibody. Finally, to clarify the source of CX3CL1 into the BM of MM patients, we explored CX3CL1 expression either by CD138+ cells on a dataset of 133 MM patients at diagnosis (GSE16122) and 23 human myeloma cell lines (HMCLs) (GSE6205), or on a proprietary dataset of primary mesenchymal stromal cells (MSCs) and osteoblasts (OBs) of 16 MM and 7 HD. Isolated BM endothelial cells from 4 MM patients and controls were also analysed. Purified CD138+ cells, HMCLs and BM endothelial cells were negative for CX3CL1 mRNA expression whereas MSCs and OBs were positive for CX3CL1 even if any statistically significant difference was not observed across the different groups. On the other hand, we found that CD138+ cells and HMCLs expressed both ADAM10 and ADAM17, leading to the hypothesis that the expression of these metalloproteinases by MM cells is accountable for the increased levels of CX3CL1 into the BM microenvironment and consequently increase of the neo-vascularization.In conclusion, our data indicate that CX3CL1, present at high level into the BM of MM patients, is involved in MM-induced BM neo-vascularization and potentially in MM progression. DisclosuresNo relevant conflicts of interest to declare.

Abstract P-41: Contribution of Matrix-bound Vesicles Produced by Mesenchymal Stromal Cells in the Differentiation of Multipotent Stem Cells in vitro

Background: According to the current view on the extracellular matrix (ECM) composition and functions, it includes not only structural proteins and components of cell adhesion, but also various deposited components, including enzymes involved in ECM remodeling, growth factors, and matrix-bound vesicles (MBV). MBV can presumably participate in the formation of a specific microenvironment for stem cells and regulate their differentiation. However, the contribution of MBV to these processes remains poorly understood. In our work, we evaluated the effects of MBV within native ECM produced by mesenchymal stromal cells (MSCs) cultured in cell sheet on multipotent stem cell differentiation. Methods: We isolated MBV from decellularized MSC-produced ECM by treatment with the following enzymes: collagenase, hyaluronidase, or trypsin, and centrifugation on 1000 kDa filters. The nanostructure and relative size in each sample were observed using TEM. The particle size and concentration were also studied with NTA. In addition, the obtained MBV were examined for the presence of key exosome markers using Western blot. Then we investigated the effect of MBV on the formation of capillary-like structures by endothelial cells (in vitro model of angiogenesis) as well as on the differentiation of primary MSCs isolated from human adipose tissue in the adipogenic, osteogenic, and chondrogenic directions. Results: As a result of comparative analysis of isolation protocols, it was shown that all MBV samples had the characteristics of extracellular vesicles (EV), but differed in size and representation of exosomal markers. The MBV isolated from ECM did not stimulate the formation of capillary-like structures by endothelial cells, in contrast to EV secreted by MSCs to the conditioned medium, but maintained the viability of the endothelium. Isolated MBV stimulated osteogenic and adipogenic differentiation of MSCs similar to secreted EV. On the other hand, preincubation of MSCs with MBV leads to reorganization of cell monolayer to spheroid-like structures during chondrogenic differentiation. Conclusion: Here, we developed the protocol of isolation of MBV from ECM that have distinguished characteristics and functional activity.

Rifampicin induces the bone form of alkaline phosphatase in humans.

Pregnane X receptor (PXR) is a xenobiotic-sensing nuclear receptor that regulates drug metabolism in the liver and intestine. In our clinical trials on healthy volunteers to discover novel metabolic functions of PXR activation, we observed that rifampicin, a well-established ligand for human PXR, 600mg daily for a week, increased the plasma alkaline phosphatase (ALP) significantly compared with the placebo. Further analysis with lectin affinity electrophoresis revealed that especially the bone form of ALP was elevated. To investigate the mechanism(s) of bone ALP induction, we employed osteoblast lineage differentiated from human primary bone marrow-derived mesenchymal stromal cells. Rifampicin treatment increased ALP activity and mRNA level of bone biomarker genes (ALP, MGP, OPN and OPG). PXR expression was detected in the cells, but the expression was very low compared with the human liver. To further investigate the potential role of PXR in the ALP induction, we treated mice and rats with a rodent PXR ligand pregnenolone 16α-carbonitrile (PCN). However, PCN treatment did not increase plasma ALP activity or bone ALP mRNA expression. In conclusion, rifampicin treatment induces the bone form of ALP in the serum of healthy human volunteers. Further studies are required to establish the mechanism of this novel finding.

Dickkopf (Dkk)-2 is a beige fat-enriched adipokine to regulate adipogenesis

In the past decades, remarkable efforts have been made to unravel the regulation of adipose tissue metabolism, given the increasing prevalence of obesity and its huge impact on human health. Wnt signaling pathway is closely involved in this entity. As extracellular inhibitors to Wnt signaling, secreted protein Dickkopfs (Dkks) may be potential targets to combat obesity and related metabolic disorders. In this study, we showed that Dkk2 was a beige fat-enriched adipokine to regulate adipogenesis. Dkk2 was strikingly expressed in beige fat depot compared to classic white, brown, and subcutaneous fat. Dkk2 treatment inhibited adipogenesis in 3T3-L1 pre-adipocytes, C3H10T1/2 mesenchymal stem cells, and primary bone marrow mesenchymal stromal cells. Activation of the master adipogenic factor PPARγ by the synthetic Thiazolidinedione ligand rosiglitazone largely rescued the inhibition of adipogenesis by Dkk2. Furthermore, adenoviral overexpression of Dkk2 in the liver to mimic its gain-of-function showed minimal effect on whole-body metabolism. These results collectively suggest that Dkk2 is a first-in-class beige fat adipokine and functions mainly through a paracrine manner to inhibit adipogenesis rather than as an endocrine factor. Our findings aid a better understanding of beige fat function and regulation and further, provide a potential therapeutic target for treating obesity.

Biomolecular phenotyping and heterogeneity assessment of mesenchymal stromal cells using label-free Raman spectroscopy

Easy, quantitative measures of biomolecular heterogeneity and high-stratified phenotyping are needed to identify and characterise complex disease processes at the single-cell level, as well as to predict cell fate. Here, we demonstrate how Raman spectroscopy can be used in the difficult-to-assess case of clonal, bone-derived mesenchymal stromal cells (MSCs) to identify MSC lines and group these according to biological function (e.g., differentiation capacity). Biomolecular stratification is achieved using high-precision measures obtained from representative statistical sampling that also enable quantified heterogeneity assessment. Application to primary MSCs and human dermal fibroblasts shows use of these measures as a label-free assay to classify cell sub-types within complex heterogeneous cell populations, thus demonstrating the potential for therapeutic translation, and broad application to the phenotypic characterisation of other cells.

Endothelium-derived stromal cells contribute to hematopoietic bone marrow niche formation

Bone marrow stromal cells (BMSCs) play pivotal roles in tissue maintenance and regeneration. Their origins, however, remain incompletely understood. Here we identify rare LNGFR+ cells in human fetal and regenerative bone marrow that co-express endothelial and stromal markers. This endothelial subpopulation displays transcriptional reprogramming consistent with endothelial-to-mesenchymal transition (EndoMT) and can generate multipotent stromal cells that reconstitute the bone marrow (BM) niche upon transplantation. Single-cell transcriptomics and lineage tracing in mice confirm robust and sustained contributions of EndoMT to bone precursor and hematopoietic niche pools. Interleukin-33 (IL-33) is overexpressed in subsets of EndoMT cells and drives this conversion process through ST2 receptor signaling. These data reveal generation of tissue-forming BMSCs from mouse and human endothelial cells and may be instructive for approaches to human tissue regeneration.

Collagen-VI Supplementation by Cell Transplantation Improves Muscle Regeneration in Ullrich Congenital Muscular Dystrophy Model Mice

Type 6 collagen (COL6) is produced by mesenchymal stromal cells (MSC) in skeletal muscle, and its gene mutation is the cause of Ullrich congenital muscular dystrophy (UCMD). To test whether COL6 supplementation has a therapeutic effect for UCMD, we conducted in vivo and in vitro experiments using healthy induced pluripotent stem cell (iPSC)-derived MSCs (iMSCs) and COL6-deficient iMSCs (KO-iMSCs). The transplantation of these iMSCs or primary MSCs into the skeletal muscle of immunodeficient UCMD model mice demonstrated that muscle regeneration and maturation were promoted only in the region supplemented with COL6. In addition, co-culture experiments of skeletal muscle satellite cells derived from UCMD model mice (Col6a1KO-MuSCs) showed iMSCs but not KO-iMSCs could improve the proliferation, differentiation, and maturation of Col6a1KO-MuSCs. These findings indicate that COL6 supplementation improves muscle regeneration and maturation in UCMD model mice and will help elucidate the pathology of UCMD and establishment of effective treatments.

Single-cell RNA sequencing of equine mesenchymal stromal cells from primary donor-matched tissue sources reveals functional heterogeneity in immune modulation and cell motility

BackgroundThe efficacy of mesenchymal stromal cell (MSC) therapy is thought to depend on the intrinsic heterogeneity of MSC cultures isolated from different tissue sources as well as individual MSCs isolated from the same tissue source, neither of which is well understood. To study this, we used MSC cultures isolated from horses. The horse is recognized as a physiologically relevant large animal model appropriate for translational MSC studies. Moreover, due to its large size the horse allows for the simultaneous collection of adequate samples from multiple tissues of the same animal, and thus, for the unique collection of donor matched MSC cultures from different sources. The latter is much more challenging in mice and humans due to body size and ethical constraints, respectively.MethodsIn the present study, we performed single-cell RNA sequencing (scRNA-seq) on primary equine MSCs that were collected from three donor-matched tissue sources; adipose tissue (AT), bone marrow (BM), and peripheral blood (PB). Based on transcriptional differences detected with scRNA-seq, we performed functional experiments to examine motility and immune regulatory function in distinct MSC populations.ResultsWe observed both inter- and intra-source heterogeneity across the three sources of equine MSCs. Functional experiments demonstrated that transcriptional differences correspond with phenotypic variance in cellular motility and immune regulatory function. Specifically, we found that (i) differential expression of junctional adhesion molecule 2 (JAM2) between MSC cultures from the three donor-matched tissue sources translated into altered cell motility of BM-derived MSCs when RNA interference was used to knock down this gene, and (ii) differences in C-X-C motif chemokine ligand 6 (CXCL6) expression in clonal MSC lines derived from the same tissue source correlated with the chemoattractive capacity of PB-derived MSCs.ConclusionsUltimately, these findings will enhance our understanding of MSC heterogeneity and will lead to improvements in the therapeutic potential of MSCs, accelerating the transition from bench to bedside.

Preclinical In Vitro Assessment of Submicron-Scale Laser Surface Texturing on Ti6Al4V.

Loosening of orthodontic and orthopedic implants is a critical and common clinical problem. To minimize the numbers of revision surgeries due to peri-implant inflammation or insufficient osseointegration, developments of new implant manufacturing strategies are indicated. Ultrafast laser surface texturing is a promising contact-free technology to modify the physicochemical properties of surfaces toward an anti-infectious functionalization. This work aims to texture Ti6Al4V surfaces with ultraviolet (UV) and green (GR) radiation for the manufacturing of laser-induced periodic surface structures (LIPSS). The assessment of these surface modifications addresses key aspects of topography, morphology and chemical composition. Human primary mesenchymal stromal cells (hMSCs) were cultured on laser-textured and polished Ti6Al4V to characterize the surfaces in terms of their in vitro biocompatibility, cytotoxicity, and metal release. The outcomes of the in vitro experiment show the successful culture of hMSCs on textured Ti6Al4V surfaces developed within this work. Cells cultured on LIPSS surfaces were not compromised in terms of their viability if compared to polished surfaces. Yet, the hMSC culture on UV-LIPSS show significantly lower lactate dehydrogenase and titanium release into the supernatant compared to polished. Thus, the presented surface modification can be a promising approach for future applications in orthodontics and orthopedics.

B-Cell Precursor Acute Lymphoblastic Leukemia Instructs Mesenchymal Stromal Cells to Alter Interferon-Related Gene Expression and Cyto/Chemokine Secretion

Introduction: B-cell precursor acute lymphoblastic leukemia (BCP-ALL) cells that are present in the bone marrow microenvironment are able to hijack the normal hematopoietic stem cell niches to create a leukemic niche. The importance of this microenvironment for leukemic cells is demonstrated by the protection that the niche provides against chemotherapeutic agents. Patient-derived mesenchymal stromal cells (MSCs) mimic this protective effect in vitro. Unraveling the mechanism of protection is important to provide potential novel options for therapeutic intervention. Therefore, our research is focused on the interaction between BCP-ALL cells and MSCs. We aimed to determine gene expression changes in BCP-ALL cells and MSCs after co-culture compared to mono-culture, and to investigate which cyto- and chemokines are differentially secreted upon contact between BCP-ALL cells and MSCs. Methods: We performed co-cultures of primary MSCs and BCP-ALL cells, and mono-cultures of MSCs or BCP-ALL cells for 40 hours to determine gene expression changes. Viable cells were sorted by FACS and RNA was isolated. Total-RNA sequencing data (Illumina) were analyzed using R. Supernatant was saved to determine cyto/chemokine profiles by Luminex technology, and to investigate the effect of these cyto/chemokines on the survival and migration of the leukemic cells. Moreover, migration experiments using transwells (3.0µm pore size) were performed to determine the level of BCP-ALL migration towards cyto/chemokines of interest. Results: RNA sequencing data from 15 independent co-culture experiments revealed that interferon (IFN)-related genes, such as IFI6, MX1, IFI27, and OAS1, were 2.5 to 3.1-fold upregulated in the MSCs after co-culture with BCP-ALL compared to MSC mono-culture. The type of upregulated pro-inflammatory genes and amount of upregulation varied between BCP-ALL patients. However, the observed changes were always similar when an ALL case was co-cultured with different MSC samples. This suggests that the observed changes are induced by the leukemic cells and that leukemic cells manipulate MSCs. Survival benefit (0.3 - 37.9%) was observed in BCP-ALL cells after co-culture with MSCs compared to BCP-ALL mono-culture. Moreover, pro-inflammatory cytokines, and several migration-related chemokines such as CCL2, CXCL8, and CXCL10/IP-10 were upregulated in 5 out of 15 co-cultures compared to the sum of the separate mono-cultures of BCP-ALL and MSCs. A gradient of CXCL10/IP-10 in transwell experiments showed that this chemokine did not enhance the migration of primary BCP-ALL cells, suggesting that the ALL-induced secretion of this chemokine serves a different role in BCP-ALL. The role of CXCL10/IP-10 and other cyto/chemokines in immune regulation at the time of overt leukemia is part of ongoing studies. Conclusion: Our data show that IFN-related genes, pro-inflammatory cytokines and migration-related chemokines become upregulated in bone marrow stromal cells upon exposure to BCP-ALL. These induced changes may be important for BCP-ALL cell survival, affecting the mobility of other immune cells, and/or ensure that leukemic cells remain in close contact with MSCs. We postulate that interference with these affected genes and cyto/chemokines may disrupt the direct contact between leukemic cells and their niche, and may provide an alternative way to eliminate leukemic cells more efficaciously. Functional studies addressing this concept are currently being executed and the results will be presented during the meeting. Disclosures No relevant conflicts of interest to declare.

Identification and characterization of a large source of primary mesenchymal stem cells tightly adhered to bone surfaces of human vertebral body marrow cavities

BackgroundTherapeutic allogeneic mesenchymal stromal cells (MSCs) are currently in clinical trials to evaluate their effectiveness in treating many different disease indications. Eventual commercialization for broad distribution will require further improvements in manufacturing processes to economically manufacture MSCs at scales sufficient to satisfy projected demands. A key contributor to the present high cost of goods sold for MSC manufacturing is the need to create master cell banks from multiple donors, which leads to variability in large-scale manufacturing runs. Therefore, the availability of large single donor depots of primary MSCs would greatly benefit the cell therapy market by reducing costs associated with manufacturing. MethodsWe have discovered that an abundant population of cells possessing all the hallmarks of MSCs is tightly associated with the vertebral body (VB) bone matrix and only liberated by proteolytic digestion. Here we demonstrate that these vertebral bone-adherent (vBA) MSCs possess all the International Society of Cell and Gene Therapy-defined characteristics (e.g., plastic adherence, surface marker expression and trilineage differentiation) of MSCs, and we have therefore termed them vBA-MSCs to distinguish this population from loosely associated MSCs recovered through aspiration or rinsing of the bone marrow compartment. ResultsPilot banking and expansion were performed with vBA-MSCs obtained from 3 deceased donors, and it was demonstrated that bank sizes averaging 2.9 × 108 ± 1.35 × 108 vBA-MSCs at passage 1 were obtainable from only 5 g of digested VB bone fragments. Each bank of cells demonstrated robust proliferation through a total of 9 passages, without significant reduction in population doubling times. The theoretical total cell yield from the entire amount of bone fragments (approximately 300 g) from each donor with limited expansion through 4 passages is 100 trillion (1 × 1014) vBA-MSCs, equating to over 105 doses at 10 × 106 cells/kg for an average 70-kg recipient. DiscussionThus, we have established a novel and plentiful source of MSCs that will benefit the cell therapy market by overcoming manufacturing and regulatory inefficiencies due to donor-to-donor variability.

Multifunctional biomimetic hydrogel systems to boost the immunomodulatory potential of mesenchymal stromal cells

Mesenchymal stromal cells (MSCs) hold great therapeutic potential, in part because of their immunomodulatory properties. However, these properties can be transient and depend on multiple factors. Here, we developed a multifunctional hydrogel system to synergistically enhance the immunomodulatory properties of MSCs, using a combination of sustained inflammatory licensing and three-dimensional (3D) encapsulation in hydrogels with tunable mechanical properties. The immunomodulatory extracellular matrix hydrogels (iECM) consist of an interpenetrating network of click functionalized-alginate and fibrillar collagen, in which interferon γ (IFN-γ) loaded heparin-coated beads are incorporated. The 3D microenvironment significantly enhanced the expression of a wide panel of pivotal immunomodulatory genes in bone marrow-derived primary human MSCs (hMSCs), compared to two-dimensional (2D) tissue culture. Moreover, the inclusion of IFN-γ loaded heparin-coated beads prolonged the expression of key regulatory genes upregulated upon licensing, including indoleamine 2,3-dioxygenase 1 (IDO1) and galectin-9 (GAL9). At a protein level, iECM hydrogels enhanced the secretion of the licensing responsive factor Gal-9 by hMSCs. Its presence in hydrogel conditioned media confirmed the correct release and diffusion of the factors secreted by hMSCs from the system. Furthermore, co-culture of iECM-encapsulated hMSCs and activated human T cells resulted in suppressed proliferation, demonstrating direct regulation on immune cells. These data highlight the potential of iECM hydrogels to enhance the immunomodulatory properties of hMSCs in cell therapies.

Immortalization of Mesenchymal Stromal Cells by TERT Affects Adenosine Metabolism and Impairs their Immunosuppressive Capacity.

Mesenchymal stromal cells (MSCs) are promising candidates for cell-based therapies, mainly due to their unique biological properties such as multipotency, self-renewal and trophic/immunomodulatory effects. However, clinical use has proven complex due to limitations such as high variability of MSCs preparations and high number of cells required for therapies. These challenges could be circumvented with cell immortalization through genetic manipulation, and although many studies show that such approaches are safe, little is known about changes in other biological properties and functions of MSCs. In this study, we evaluated the impact of MSCs immortalization with the TERT gene on the purinergic system, which has emerged as a key modulator in a wide variety of pathophysiological conditions. After cell immortalization, MSCs-TERT displayed similar immunophenotypic profile and differentiation potential to primary MSCs. However, analysis of gene and protein expression exposed important alterations in the purinergic signaling of in vitro cultured MSCs-TERT. Immortalized cells upregulated the CD39/NTPDase1 enzyme and downregulated CD73/NT5E and adenosine deaminase (ADA), which had a direct impact on their nucleotide/nucleoside metabolism profile. Despite these alterations, adenosine did not accumulate in the extracellular space, due to increased uptake. MSCs-TERT cells presented an impaired in vitro immunosuppressive potential, as observed in an assay of co-culture with lymphocytes. Therefore, our data suggest that MSCs-TERT have altered expression of key enzymes of the extracellular nucleotides/nucleoside control, which altered key characteristics of these cells and can potentially change their therapeutic effects in tissue engineering in regenerative medicine.

Transplantation of Mesenchymal Stromal Cells Expressing the Human Preproenkephalin Gene Can Relieve Pain in a Rat Model of Neuropathic Pain.

Transgenic therapy for central neuralgia faces the problems of low expression and weak targeting and affects superficial but not deep neurons. In this study, we generated a lentivirus vector with human preproenkephalin gene (hPPE) expression driven by the transcriptional amplification strategy system (TAS) and established a primary bone marrow-derived mesenchymal stromal cell (BMSC) line stably expressing hPPE for transplantation into a rat model of neuropathic pain rat. The paw thermal withdrawal latency assay and paw mechanical withdrawal threshold assay showed that unlike control BMSCs and BMSCs with hPPE overexpression driven by the CMV or Synapsin 1 (SYN1) promoter, TAS-hPPE BMSCs had a robust and lasting analgesic effect. The TAS-hPPE BMSC-treated group exhibited higher expression of TAS-driven hPPE and a higher ratio of BMSCs in the midbrain, spinal cord and cortex then the CMV-hPPE BMSC- and SYN1-hPPE BMSC-treated groups. Moreover, we also observed that TAS-hPPE BMSCs displayed a greater tendency to differentiate into neurons and exhibit neuronal-like distribution than CMV-hPPE or SYN1-hPPE BMSCs. In conclusion, our study shows that the TAS improves BMSC transgenic therapy for neuropathic pain treatment.

Abstract B24: A multicellular, microfluidic model of the human bone marrow niche in metastatic cancers

Abstract Bone metastases cause significant morbidity in solid tumors, with prostate, breast, lung, and kidney cancers representing the most common primary sources. Understanding the landscape of stromal interactions in bone and bone marrow would facilitate development of novel therapies targeting this unique metastatic niche. Mesenchymal stromal cells (MSCs) are multipotent cells in bone marrow that differentiate into osteoblasts, adipocytes, and fibroblasts and regulate immune and inflammatory responses. MSC recruitment into primary tumors enhances invasiveness of prostate adenocarcinoma, which suggests that bone marrow MSCs impact development of bone metastases in prostate cancer and promote treatment resistance. We hypothesize that multicellular interactions drive a permissive bone marrow environment for outgrowth of metastatic lesions. The goal of this study is to develop a multicellular bone stromal model from patients with solid tumors for functional and molecular analysis of cellular crosstalk. We have recently published a reconfigurable, microfluidic platform that enables culture of up to 6 different cell types in 3-dimensional matrices and multi-endpoint analysis of limited patient samples. In this study, we have modified this platform to culture patient-derived tissue, by using primary bone marrow cells from patients consented to provide bone marrow aspirate at time of radical prostatectomy. This technology allows spatiotemporal control of individual treatment conditions, enabling culture and differentiation of primary human cells in separate chips that can be reconfigured into defined microenvironments. Subsequently, specific compartments can be isolated for gene expression analysis and in-device imaging. Using this platform, we present two distinct bone marrow models. In the first model, we cultured primary human bone marrow MSCs that were induced into osteoblastic and adipocytic lineages in 3D microculture. MSCs induced invasion of prostate cancer cell lines, and paracrine crosstalk between MSCs and prostate cancer cells increased expression of IL-6, IL-8, CXCL12, and MMP-9 in MSCs. In the second model, we generated a multicellular mixed 3D culture in which osteoblasts and adipocytes spontaneously differentiate in coculture with stromal cells, osteoclasts, and leukocytes. The mixed 3D cultures generated spheroid structures that resemble stem cell components with self-renewal capability, previously called mesenspheres. Overall, we have recapitulated cellular compartments in the bone marrow niche using two complementary methods. The impact of defined molecular subtypes of cancer is now being tested to understand if specific genetic alterations promote development of invasive cancer in bone marrow. Future directions include analysis of paracrine signaling and functional interactions between disseminated cancer cells and bone marrow cellular stroma as potential therapeutic targets. Citation Format: Nan Sethakorn, David Kosoff, Erika Heninger, Jasmine Martinez Soto, Jiaquan Yu, Rahul Das, David Jarrard, Jacques Galipeau, Peiman Hematti, Scott Dehm, David J. Beebe, Joshua M. Lang. A multicellular, microfluidic model of the human bone marrow niche in metastatic cancers [abstract]. In: Proceedings of the AACR Special Conference on the Evolving Landscape of Cancer Modeling; 2020 Mar 2-5; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2020;80(11 Suppl):Abstract nr B24.

Genetic barcoding reveals clonal dominance in iPSC-derived mesenchymal stromal cells

BackgroundThe use of mesenchymal stromal cells (MSCs) for research and clinical application is hampered by cellular heterogeneity and replicative senescence. Generation of MSC-like cells from induced pluripotent stem cells (iPSCs) may circumvent these limitations, and such iPSC-derived MSCs (iMSCs) are already tested in clinical trials. So far, a comparison of MSCs and iMSCs was particularly addressed in bulk culture. Despite the high hopes in cellular therapy, only little is known how the composition of different subclones changes in these cell preparations during culture expansion.MethodsIn this study, we used multicolor lentiviral genetic barcoding for the marking of individual cells within cell preparations. Based on this, we could track the clonal composition of syngenic MSCs, iPSCs, and iMSCs during culture expansion. Furthermore, we analyzed DNA methylation patterns at senescence-associated genomic regions by barcoded bisulfite amplicon sequencing. The proliferation and differentiation capacities of individual subclones within MSCs and iMSCs were investigated with limiting dilution assays.ResultsOverall, the clonal composition of primary MSCs and iPSCs gradually declined during expansion. In contrast, iMSCs became oligoclonal early during differentiation, indicating that they were derived from few individual iPSCs. This dominant clonal outgrowth of iMSCs was not associated with changes in chromosomal copy number variation. Furthermore, clonal dynamics were not clearly reflected by stochastically acquired DNA methylation patterns. Limiting dilution assays revealed that iMSCs are heterogeneous in colony formation and in vitro differentiation potential, while this was even more pronounced in primary MSCs.ConclusionsOur results indicate that the subclonal diversity of MSCs and iPSCs declines gradually during in vitro culture, whereas derivation of iMSCs may stem from few individual iPSCs. Differentiation regimen needs to be further optimized to achieve homogeneous differentiation of iPSCs towards iMSCs.

Senescence-Associated Metabolomic Phenotype in Primary and iPSC-Derived Mesenchymal Stromal Cells.

SummaryLong-term culture of primary cells is characterized by functional and secretory changes, which ultimately result in replicative senescence. It is largely unclear how the metabolome of cells changes during replicative senescence and if such changes are consistent across different cell types. We have directly compared culture expansion of primary mesenchymal stromal cells (MSCs) and induced pluripotent stem cell-derived MSCs (iMSCs) until they reached growth arrest. Both cell types acquired similar changes in morphology, in vitro differentiation potential, senescence-associated β-galactosidase, and DNA methylation. Furthermore, MSCs and iMSCs revealed overlapping gene expression changes, particularly in functional categories related to metabolic processes. We subsequently compared the metabolomes of MSCs and iMSCs and observed overlapping senescence-associated changes in both cell types, including downregulation of nicotinamide ribonucleotide and upregulation of orotic acid. Taken together, replicative senescence is associated with a highly reproducible senescence-associated metabolomics phenotype, which may be used to monitor the state of cellular aging.