Bone Marrow-derived Stromal Cells Research Articles

Local SDF-1α application enhances the therapeutic efficacy of BMSCs transplantation in osteoporotic bone healing

Bone defect healing is markedly impaired in osteoporotic patient due to poor bone regeneration ability. Stromal cell derived factor-1α (SDF-1α) plays a pivotal role in the repair of various injured tissues including bone. Here, we definite that SDF-1α hydrogels potentiates in vivo osteogenesis of bone marrow-derived stromal stem cells (BMSCs) in osteoporosis. The characteristics of rat primary BMSCs including superficial markers by flow cytometry and multi-lineage differentiation by induction were determined. At different time intervals, the release media from the SDF-1α-releasing hydrogels were collected to identificate SDF-1α exhibited a sustained release profile and maintained its bioactivity after release from the hydrogels to stimulate chemotaxis of BMSCs in a time dependent manner. Bilateral alveolar defects were operated in ovariectomized (OVX) rats and repaired with systemic BMSCs transplantation with or without the hydrogels. Local administration of SDF-1α significantly enhanced BMSCs recruitment and promoted more bone regeneration as well as the expression of OCN and Runx2 compared with the effect of BMSCs transplantation alone. Moreover, after BMSCs transplantation with SDF-1α delivery, macrophage polarization was promoted toward the M2 phenotype, that is identified as an important symbol in tissue regeneration process. Taken together, local SDF-1α application enhances the efficacy of BMSCs transplantation therapy in osteoporotic bone healing, suggesting clinical potential of SDF-1α to serve as a therapeutic drug target for osteoporosis treatment.

Reinforcing interpenetrating network hydrogels with 3D printed polymer networks to engineer cartilage mimetic composites

Engineering constructs that mimic the complex structure, composition and biomechanics of the articular cartilage represents a promising route to joint regeneration. Such tissue engineering strategies require the development of biomaterials that mimic the mechanical properties of articular cartilage whilst simultaneously providing an environment supportive of chondrogenesis. Here three-dimensional (3D) bioprinting is used to develop polycaprolactone (PCL) fibre networks to mechanically reinforce interpenetrating network (IPN) hydrogels consisting of alginate and gelatin methacryloyl (GelMA). Inspired by the significant tension-compression nonlinearity of the collagen network in articular cartilage, we printed reinforcing PCL networks with different ratios of tensile to compressive modulus. Synergistic increases in compressive modulus were observed when IPN hydrogels were reinforced with PCL networks that were relatively soft in compression and stiff in tension. The resulting composites possessed equilibrium and dynamic mechanical properties that matched or approached that of native articular cartilage. Finite Element (FE) modelling revealed that the reinforcement of IPN hydrogels with specific PCL networks limited radial expansion and increased the hydrostatic pressure generated within the IPN upon the application of compressive loading. Next, multiple-tool biofabrication techniques were used to 3D bioprint PCL reinforced IPN hydrogels laden with a co-culture of bone marrow-derived stromal cells (BMSCs) and chondrocytes (CCs). The bioprinted biomimetic composites were found to support robust chondrogenesis, with encapsulated cells producing hyaline-like cartilage that stained strongly for sGAG and type II collagen deposition, and negatively for type X collagen and calcium deposition. Taken together, these results demonstrate how 3D bioprinting can be used to engineer constructs that are both pro-chondrogenic and biomimetic of the mechanical properties of articular cartilage.

Dexras1 plays a pivotal role in maintaining the equilibrium between adipogenesis and osteogenesis

BackgroundChronic steroid treatment causes an increase in visceral adiposity and osteoporosis. It is believed that steroids may alter a balance between differentiation of mesenchymal stem cells (MSCs) into either adipocytes or osteoblasts; however, the detailed molecular mechanisms are unclear. We previously identified Dexras1 as a critical factor that potentiates adipogenesis in response to glucocorticoids. Thus, in this study, we investigated the role of Dexras1 in maintaining the balance between chronic steroid treatment-associated adipogenesis and osteoporosis. Material and methodsWe treated wild type (WT) and Dexras1 knockout (KO) mice with dexamethasone for five weeks followed by 60% HFD for additional two weeks with dexamethasone. The changes of glucocorticoid-induced body weight gain and osteoporosis were analyzed. Bone marrow derived stromal cells (BMSCs) and mouse embryonic fibroblasts (MEFs) extracted from WT and Dexras1 KO mice, as well as MC3T3-E1 pre-osteoblasts and osteoclasts differentiated from RAW264.7 were analyzed to further define the role of Dexras1 in osteoblasts and osteoclasts. ResultsDual-energy X-ray absorptiometry and micro-computed tomography analyses in murine femurs revealed that Dexras1 deficiency was associated with increased osteogenesis, concurrent with reduced adipogenesis. Furthermore, Dexras1 deficiency promoted osteogenesis of BMSCs and MEFs in vitro, suggesting that Dexras1 deficiency prevents steroid-induced osteoporosis. We also observed that Dexras1 downregulated SMAD signaling pathways, which reduced the osteogenic differentiation capacity of pre-osteoblast MC3T3-E1 cells into mature osteoblasts. ConclusionWe propose that Dexras1 is critical for maintaining the equilibrium between adipogenesis and osteogenesis upon steroid treatment.

Surface modifications to polydimethylsiloxane substrate for stabilizing prolonged bone marrow stromal cell culture

Polydimethylsiloxane (PDMS) has been extensively used as a supporting material for studies of cell mechanobiology, cell micropatterning and microscale-cell analysis in microfluidic chips due to its numerous advantages, such as low cytotoxicity, ease of modification, inexpensive costs and biocompatibility. However, the innate hydrophobicity of PDMS often poses a problem for stable cell adhesion, seriously limiting its applicability for prolonged cell culture. UV exposure and protein coating are suboptimal solutions, while chemical surface functionalization is often associated with laborious procedures and producing environmental toxics. Plasma treatment can render a hydrophilic substrate by altering the surface chemistry, but such effect is often short-lived due to its tendency to hydrophobic recovery. Variation of physical properties of the substratum are known to influence cell behaviour. Nevertheless, the combination of varying PDMS substratum properties via base:curing agent ratio and plasma treatment to stabilize the long-term culture of bone marrow derived stromal cells (BMSCs) still remain poorly understood. In this study, we developed a protocol to maintain the hydrophilicity of the plasma-treated PDMS over a range of substratum properties. This study demonstrated that varying the substratum properties of PDMS can enhance the stability of BMSC culture for at least three weeks, while plasma treatment with or without additional collagen coating further enhanced such effect. The changes in the physical properties of PDMS have rendered difference in BMSCs adhesion, proliferation and in-vitro plasticity, thereby offering a simple and effective strategy for PDMS surface modification to enable long term cell analysis in PDMS-based culture platform.

Characterisation and evaluation of the regenerative capacity of Stro-4+ enriched bone marrow mesenchymal stromal cells using bovine extracellular matrix hydrogel and a novel biocompatible melt electro-written medical-grade polycaprolactone scaffold

Many skeletal tissue regenerative strategies centre around the multifunctional properties of bone marrow derived stromal cells (BMSC) or mesenchymal stem/stromal cells (MSC)/bone marrow derived skeletal stem cells (SSC). Specific identification of these particular stem cells has been inconclusive. However, enriching these heterogeneous bone marrow cell populations with characterised skeletal progenitor markers has been a contributing factor in successful skeletal bone regeneration and repair strategies. In the current studies we have isolated, characterised and enriched ovine bone marrow mesenchymal stromal cells (oBMSCs) using a specific antibody, Stro-4, examined their multipotential differentiation capacity and, in translational studies combined Stro-4+ oBMSCs with a bovine extracellular matrix (bECM) hydrogel and a biocompatible melt electro-written medical-grade polycaprolactone scaffold, and tested their bone regenerative capacity in a small in vivo, highly vascularised, chick chorioallantoic membrane (CAM) model and a preclinical, critical-sized ovine segmental tibial defect model.Proliferation rates and CFU-F formation were similar between unselected and Stro-4+ oBMSCs. Col1A1, Col2A1, mSOX-9, PPARG gene expression were upregulated in respective osteogenic, chondrogenic and adipogenic culture conditions compared to basal conditions with no significant difference between Stro-4+ and unselected oBMSCs. In contrast, proteoglycan expression, alkaline phosphatase activity and adipogenesis were significantly upregulated in the Stro-4+ cells. Furthermore, with extended cultures, the oBMSCs had a predisposition to maintain a strong chondrogenic phenotype. In the CAM model Stro-4+ oBMSCs/bECM hydrogel was able to induce bone formation at a femur fracture site compared to bECM hydrogel and control blank defect alone. Translational studies in a critical-sized ovine tibial defect showed autograft samples contained significantly more bone, (4250.63 mm3, SD = 1485.57) than blank (1045.29 mm3, SD = 219.68) ECM-hydrogel (1152.58 mm3, SD = 191.95) and Stro-4+/ECM-hydrogel (1127.95 mm3, SD = 166.44) groups.Stro-4+ oBMSCs demonstrated a potential to aid bone repair in vitro and in a small in vivo bone defect model using select scaffolds. However, critically, translation to a large related preclinical model demonstrated the complexities of bringing small scale reported stem-cell material therapies to a clinically relevant model and thus facilitate progression to the clinic.

Additive manufactured, highly resilient, elastic, and biodegradable poly(ester)urethane scaffolds with chondroinductive properties for cartilage tissue engineering.

Articular cartilage was thought to be one of the first tissues to be successfully engineered. Despite the avascular and non-innervated nature of the tissue, the cells within articular cartilage – chondrocytes – account for a complex phenotype that is difficult to be maintained in vitro. The use of bone marrow–derived stromal cells (BMSCs) has emerged as a potential solution to this issue. Differentiation of BMSCs toward stable and non-hypertrophic chondrogenic phenotypes has also proved to be challenging. Moreover, hyaline cartilage presents a set of mechanical properties – relatively high Young's modulus, elasticity, and resilience – that are difficult to reproduce. Here, we report on the use of additive manufactured biodegradable poly(ester)urethane (PEU) scaffolds of two different structures (500 μm pore size and 90° or 60° deposition angle) that can support the loads applied onto the knee while being highly resilient, with a permanent deformation lower than 1% after 10 compression-relaxation cycles. Moreover, these scaffolds appear to promote BMSC differentiation, as shown by the deposition of glycosaminoglycans and collagens (in particular collagen II). At gene level, BMSCs showed an upregulation of chondrogenic markers, such as collagen II and the Sox trio, to higher or similar levels than that of traditional pellet cultures, with a collagen II/collagen I relative expression of 2–3, depending on the structure of the scaffold. Moreover, scaffolds with different pore architectures influenced the differentiation process and the final BMSC phenotype. These data suggest that additive manufactured PEU scaffolds could be good candidates for cartilage tissue regeneration in combination with microfracture interventions.

Treatment of rats with spinal cord injury using human bone marrow-derived stromal cells prepared by negative selection

BackgroundSpinal cord injury (SCI) is a highly debilitating pathology without curative treatment. One of the most promising disease modifying strategies consists in the implantation of stem cells to reduce inflammation and promote neural regeneration. In the present study we tested a new human bone marrow-derived stromal cell preparation (bmSC) as a therapy of SCI.MethodsSpinal cord contusion injury was induced in adult male rats at thoracic level T9/T10 using the Infinite Horizon impactor. One hour after lesion the animals were treated with a sub-occipital injection of human bmSC into the cisterna magna. No immune suppression was used. One dose of bmSC consisted, on average, of 2.3 million non-manipulated cells in 100 μL suspension, which was processed out of fresh human bone marrow from the iliac crest of healthy volunteers. Treatment efficacy was compared with intraperitoneal injections of methylprednisolone (MP) and saline. The recovery of motor functions was assessed during a surveillance period of nine weeks. Adverse events as well as general health, weight and urodynamic functions were monitored daily. After this time, the animals were perfused, and the spinal cord tissue was investigated histologically.ResultsRats treated with bmSC did not reject the human implants and showed no sign of sickness behavior or neuropathic pain. Compared to MP treatment, animals displayed better recovery of their SCI-induced motor deficits. There were no significant differences in the recovery of bladder control between groups. Histological analysis at ten weeks after SCI revealed no differences in tissue sparing and astrogliosis, however, bmSC treatment was accompanied with reduced axonal degeneration in the dorsal ascending fiber tracts, lower Iba1-immunoreactivity (IR) close to the lesion site and reduced apoptosis in the ventral grey matter. Neuroinflammation, as evidenced by CD68-IR, was significantly reduced in the MP-treated group.ConclusionsHuman bmSC that were prepared by negative selection without expansion in culture have neuroprotective properties after SCI. Given the effect size on motor function, implantation in the acute phase was not sufficient to induce spinal cord repair. Due to their immune modulatory properties, allogeneic implants of bmSC can be used in combinatorial therapies of SCI.

Porous titanium fiber mesh with tailored elasticity and its effect on stromal cells.

Porous titanium fiber mesh (TFM) is considered a suitable scaffold material for bone reconstruction. Also, TFM can be used to cover the surface of bone‐anchored devices, that is, orthopedic or dental implants. The titanium fiber size has an effect of the stiffness as well as porosity of the titanium mesh, which can influence the behavior of bone forming cells. Therefore, the aim of this study was to vary TFM composition, in order to achieve different stiffness, and to assess the effects of such variation on the behavior of bone marrow‐derived stromal cells (BMSCs). With that purpose, nine types of TFM (porosities 60–87%; fiber size 22–50 μm), were examined for their mechanical properties as well as their effect on the proliferation and differentiation of rat bone marrow‐derived stromal cells (rBMSCs) up to 21 days. Dynamic mechanical analysis revealed that the stiffness of TFM were lower than of solid titanium and decreased with larger fiber sizes. The stiffness could effectively be tailored by altering fiber properties, which altered the pore simultaneously. For the 22 and 35 μm size fiber meshes with the highest porosity, the stiffness closely matched the value found in literature for cortical bone. Finally, all tested TFM types supported the growth and differentiation of rBMSCs. We concluded that TFM material has been proven cytocompatible. Further preclinical studies are needed to assess which TFM type is most suitable as clinical use for bone ingrowth and bone regeneration.

Enhancing the Efficacy of Stem Cell Therapy with Glycosaminoglycans.

SummaryHuman mesenchymal stem cell (hMSC) therapy offers significant potential for osteochondral regeneration. Such applications require their ex vivo expansion in media frequently supplemented with fibroblast growth factor 2 (FGF2). Particular heparan sulfate (HS) fractions stabilize FGF2-FGF receptor complexes. We show that an FGF2-binding HS variant (HS8) accelerates the expansion of freshly isolated bone marrow hMSCs without compromising their naivety. Importantly, the repair of osteochondral defects in both rats and pigs is improved after treatment with HS8-supplemented hMSCs (MSCHS8), when assessed histologically, biomechanically, or by MRI. Thus, supplementing hMSC culture media with an HS variant that targets endogenously produced FGF2 allows the elimination of exogenous growth factors that may adversely affect their therapeutic potency.

Multi-Parameter Analysis of Biobanked Human Bone Marrow Stromal Cells Shows Little Influence for Donor Age and Mild Comorbidities on Phenotypic and Functional Properties.

Heterogeneous populations of human bone marrow-derived stromal cells (BMSC) are among the most frequently tested cellular therapeutics for treating degenerative and immune disorders, which occur predominantly in the aging population. Currently, it is unclear whether advanced donor age and commonly associated comorbidities affect the properties of ex vivo-expanded BMSCs. Thus, we stratified cells from adult and elderly donors from our biobank (n = 10 and n = 13, mean age 38 and 72 years, respectively) and compared their phenotypic and functional performance, using multiple assays typically employed as minimal criteria for defining multipotent mesenchymal stromal cells (MSCs). We found that BMSCs from both cohorts meet the standard criteria for MSC, exhibiting similar morphology, growth kinetics, gene expression profiles, and pro-angiogenic and immunosuppressive potential and the capacity to differentiate toward adipogenic, chondrogenic, and osteogenic lineages. We found no substantial differences between cells from the adult and elderly cohorts. As positive controls, we studied the impact of in vitro aging and inflammatory cytokine stimulation. Both conditions clearly affected the cellular properties, independent of donor age. We conclude that in vitro aging rather than in vivo donor aging influences BMSC characteristics.

Screening differentially expressed proteins from co-cultured hematopoietic cells and bone marrow-derived stromal cells by quantitative proteomics (SILAC) method

BackgroundBone marrow stromal cells protect hematopoietic cells and provide drug resistance by delivering bunch of variable proteins. Thus, alterations of protein expression are typically associated with cell–cell signal transduction and regulation of cellular functions.MethodsCo-culture models of bone marrow stromal cells and hematopoietic cells are often used in studies of their crosstalk. Studies of altered protein expression initiated by stromal cell/hematopoietic cell interactions are an important new trend in microenvironmental research. There has been no report to date of global quantitative proteomics analysis of crosstalk between hematopoietic cells and stromal cells. In this study, we analyzed quantitative proteomes in a co-culture system of stromal HS5 cells and hematopoietic KG1a cells, and simultaneously tracked differentially expressed proteins in two types of cells before and after co-culture by stable isotope labeling by amino acids in cell culture (SILAC) method.ResultsWe have shown that in co-cultured KG1a, 40 proteins (including CKAP4, LMNA, and SERPINB2) were upregulated and 64 proteins (including CD44, CD99, and NCAM1) were downregulated relative to KG1a alone. We utilized IPA analysis to discover that the NOD-like receptor signaling pathway was upregulated, whereas platelet activation was downregulated in co-cultured KG1a cells. Furthermore, 95 proteins (including LCP1, ARHGAP4, and UNCX) were upregulated and 209 proteins (including CAPG, FLNC, and MAP4) were downregulated in co-cultured HS5 relative to HS5 alone. The tight junction pathway was downregulated and glycolysis/gluconeogenesis pathway was dysfunctional in co-cultured HS5. Most importantly, the significantly differentially expressed proteins can also be confirmed using different co-cultured cell lines.ConclusionAltogether, we recommend such quantitative proteomics approach for the studies of the hematopoietic–stroma cross-talk, differentially expressed proteins and related signaling pathways identification. The differentially expressed proteins identified from this current SILAC method will provide a useful basis for ongoing studies of crosstalk between stromal cells and hematopoietic cells in co-culture systems. All these result suggested our ongoing studies can focus on the mechanisms underlying CKAP4 increase and CD44 decrease in co-cultured hematopoietic cells, and the increase of LCP1 and decrease of CAPG in co-cultured stromal cell. The proteomic profiles from the KG1a/stromal cell co-culture system give new molecular insights into the roles of these cells in MDS pathophysiology and related bone disease.

Phenotypic Characterization of Bone Marrow Mononuclear Cells and Derived Stromal Cell Populations from Human Iliac Crest, Vertebral Body and Femoral Head.

(1) In vitro, bone marrow-derived stromal cells (BMSCs) demonstrate inter-donor phenotypic variability, which presents challenges for the development of regenerative therapies. Here, we investigated whether the frequency of putative BMSC sub-populations within the freshly isolated mononuclear cell fraction of bone marrow is phenotypically predictive for the in vitro derived stromal cell culture. (2) Vertebral body, iliac crest, and femoral head bone marrow were acquired from 33 patients (10 female and 23 male, age range 14–91). BMSC sub-populations were identified within freshly isolated mononuclear cell fractions based on cell-surface marker profiles. Stromal cells were expanded in monolayer on tissue culture plastic. Phenotypic assessment of in vitro derived cell cultures was performed by examining growth kinetics, chondrogenic, osteogenic, and adipogenic differentiation. (3) Gender, donor age, and anatomical site were neither predictive for the total yield nor the population doubling time of in vitro derived BMSC cultures. The abundance of freshly isolated progenitor sub-populations (CD45−CD34−CD73+, CD45−CD34−CD146+, NG2+CD146+) was not phenotypically predictive of derived stromal cell cultures in terms of growth kinetics nor plasticity. BMSCs derived from iliac crest and vertebral body bone marrow were more responsive to chondrogenic induction, forming superior cartilaginous tissue in vitro, compared to those isolated from femoral head. (4) The identification of discrete progenitor populations in bone marrow by current cell-surface marker profiling is not predictive for subsequently derived in vitro BMSC cultures. Overall, the iliac crest and the vertebral body offer a more reliable tissue source of stromal progenitor cells for cartilage repair strategies compared to femoral head.

Radioresistance of Serpinb3a-/- Mice and Derived Hematopoietic and Marrow Stromal Cell Lines.

Serpins are a group of serine-proteases involved in multiple signal transduction pathways in mammalian cells. In particular, Serpinb3a is involved in the lysosomal necrosis cell death pathway with components that overlap with radiation-induced apoptosis. We investigated the radiation response of Serpinb3a-/- mice compared to Serpinb3a+/+ mice on the Balb/c background. Serpinb3a-/- mice showed significant radioresistance to a dose of 8.0 Gy total-body irradiation, compared to Serpinb3a+/+ Balb/c mice. Long-term bone marrow cultures from Serpinb3a-/- mice showed increased longevity. In clonogenic survival assays, fresh bone marrow hematopoietic progenitors, as well as clonal interleukin-3 (IL-3)-dependent hematopoietic progenitor and bone marrow stromal cell lines from Serpinb3a-/- mice were radioresistant. Serpinb3a-/- mouse bone marrow-derived stromal cell lines had increased baseline and postirradiation antioxidant capacity. Serpinb3a-/- bone marrow stromal cells showed increased radiation-induced RNA transcripts for MnSOD and p21, and decreased levels of p53 and TGF-b. Both irradiated Serpinb3a-/- mouse bone marrow stromal cell lines and plasma removed from total-body irradiated mice had decreased levels of expression of stress response and inflammation-associated proteins. Abrogation of Serpinb3a may be a potential new target for mitigation of radiation effects.

Abstract 2742: Bone marrow-derived stromal cells in the tumor microenvironment might be associated with the progression of gastric cancer

Abstract Purpose: It has been reported that the interaction between cancer cells and cancer-associated fibroblasts (CAFs) is closely associated with the progression of various types of cancer. The aim of this study is to clarify the role of bone marrow-derived stromal cells (BM-SCs), which are supposed to be the origin of CAFs in the tumor microenvironment, on the development of gastric cancer. Methods: The effect of human BM-SCs which frequently express CD271 on the proliferation and motility of six gastric cancer cell lines, OCUM-2M, OCUM-2MD3, OCUM-12, KATO-III, NUGC-3, and MKN-74, was examined. CD271 expression levels in BM-SCs were analyzed by flow cytometry. We also generated a gastric tumor model by orthotopic inoculation of OCUM-2MD3 cells in mice that had received transplantation of bone marrow from CAG-EGFP mice. The correlation between the clinicopathological features of 279 primary gastric carcinomas and CD271 expression in tumor stroma was examined by immunohistochemistry. Results: Numerous BM-SCs infiltrated the gastric tumor microenvironment; CD271 expression was found approximately 25% of BM-SCs. Conditioned medium from BM-SCs significantly increased the proliferation of gastric cancer cell lines. Furthermore, conditioned medium from gastric cancer cells significantly increased the number of BM-SCs, while migration of OCUM-12 and NUGC-3 cells was significantly increased by conditioned medium from BM-SCs. CD271 expression in stromal cells was significantly associated with macroscopic type-4 cancers, diffuse-type tumors, and tumor invasion depth. The overall survival of patients (n=279) with CD271-positive stromal cells was significantly worse than that of patients with CD271-negative stromal cells. This is the first report of the significance of BM-SCs in gastric cancer progression. Conclusions: BM-SCs were abundant in the tumor microenvironment of gastric cancer in vivo. In vitro study also indicated that the crosstalk between BM-SCs and gastric cancer cells might play an important role for the development of gastric cancer. CD271-positive tumor stromal cells might be a useful predictive prognostic factor for patients with gastric cancer. BM-SCs, therefore, might be one of therapeutic targets for cancer treatment. Citation Format: Masakazu Yashiro, Hiroaki Kasashima, Syuhei Kushiyama, Sadaaki Nishimura, Kenji Kuroda, Shingo Togano, Tomohisa Okuno, Yuichiro Miki, Tatsunari Fukuoka, Hirohisa Nakamae, Masayuki Hino, Masaichi Ohira. Bone marrow-derived stromal cells in the tumor microenvironment might be associated with the progression of gastric cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2742.

Dynamic secretome of bone marrow-derived stromal cells reveals a cardioprotective biochemical cocktail

Transplanted stromal cells have demonstrated considerable promise as therapeutic agents in diverse disease settings. Paracrine signaling can be an important mediator of these therapeutic effects at the sites of acute or persistent injury and inflammation. As many stromal cell types, including bone marrow-derived stromal cells (BMSCs), display tissue-specific responses, there is a need to explore their secretory dynamics in the context of tissue and injury type. Paracrine signals are not static, and could encode contextual dynamics in the kinetic changes of the concentrations of the secreted ligands. However, precise measurement of dynamic and context-specific cellular secretory signatures, particularly in adherent cells, remains challenging. Here, by creating an experimental and computational analysis platform, we reconstructed dynamic secretory signatures of cells based on a very limited number of time points. By using this approach, we demonstrate that the secretory signatures of CD133-positive BMSCs are uniquely defined by distinct biological contexts, including signals from injured cardiac cells undergoing oxidative stress, characteristic of cardiac infarction. Furthermore, we show that the mixture of recombinant factors reproducing the dynamics of BMSC-generated secretion can mediate a highly effective rescue of cells injured by oxidative stress and an improved cardiac output. These results support the importance of the dynamic multifactorial paracrine signals in mediating remedial effects of stromal stem cells, and pave the way for stem cell-inspired cell-free treatments of cardiac and other injuries.

PS1310 THE NOVEL BISPECIFIC CD47‐CD19 ANTIBODY TG‐1801 POTENTIATES THE ACTIVITY OF UBLITUXIMAB‐UMBRALISIB (U2) DRUG COMBINATION IN PRECLINICAL MODELS OF B‐NHL

Background:Umbralisib is an orally available PI3Kδ inhibitor that has demonstrated activity both in preclinical models and primary B cell non‐Hodgkin lymphoma (B‐NHL) cells, and in patients with B‐cell malignancies. The doublet therapy of umbralisib used in combination with the glycoengineered anti‐CD20 monoclonal antibody (mAb) ublituximab (“U2” regimen), provides a non‐chemotherapy backbone regimen on which several novel multidrug combinations have been explored clinically. TG‐1801 is a novel, bispecific antibody in Phase 1 testing that targets CD47 selectively on CD19+ B‐cells, sparing red blood cells or platelets, blocking the CD47‐SIRPα macrophage checkpoint on mature B cells.Aims:We explored in vitro and in vivo potential synergies between TG‐1801 and ublituximab, umbralisib, and the U2 combination in preclinical models of B‐NHL.Methods:Human B‐cell lymphoma Raji and Jeko‐1 cells were cultured in vitro in the presence of different stromal and immune cell types including bone marrow derived stromal cells (BMSCs), M2‐polarized primary macrophages, and primary circulating PBMCs. Cells were analyzed by proliferation assay, transcriptomic analysis (qPCR array and RNA sequencing followed by gene set enrichment analysis) and fluorimetric evaluation of antibody‐dependent cell death (ADCC) and antibody‐dependent cell phagocytosis (ADCP). In vivo, drug efficacy was determined in a Raji xenograft model, by dosing tumor‐bearing mice for 17 days with ublituximab (5 mg/kg, qw) +/‐ umbralisib (150 mg/kg, bid), the combination of both and/or TG‐1801 (5 mg/kg, qw).Results:In the context of the B‐NHL co‐culture system, U2 combination treatment led to the decrease of anti‐inflammatory cytokines genes (IL‐6, IL‐10, IL‐1RA) and upregulation of pro‐inflammatory IL‐2, accompanied by a 2 fold increase of M2 dependent ADCP. When U2 was combined with TG‐1801, this 2‐fold increase in ADCP was doubled, brought to a 4‐fold increase. In addition, TG‐1801 alone activated ADCC 4.1‐fold above control, and when combined with ublituximab was 7.2‐fold above control. As expected, umbralisib did not impact ADCC. Transcriptomic analysis of CD20+ cells further revealed that U2 treatment was mainly associated with modulation of redox processes in Raji co‐cultures (NES = −0.57, p = 0.016). Although TG‐1801 single agent did not significantly affect the transcriptome of these cells, combination with U2 provoked the down‐regulation of genes associated to cell architecture homeostasis, including cellular membranes (NES = −0.57, p = 0.006), cytoskeleton (NES = −0.51, p = 0.046), and cell proliferation (NES = −0.44, p = 0.018). In vivo, ublituximab alone displayed a tumor growth inhibition (TGI) of 88%, with 3/8 mice harboring a barely palpable tumor, while the umbralisib alone treatment arm showed a TGI of 50%, with 2/8 mice lacking detectable tumors. TG‐1801 exhibited a 76% TGI with 1/8 tumor free‐mouse. Most importantly, the combination of TG‐1801 with umbralisib alone, ublituximab alone, and U2 achieved TGI of 85%, 93% and 93% respectively, with more tumor‐free mice 35 days after the last dose in these 3 groups. Interestingly, this superior anti‐tumor effect of the different TG‐1801 combinations was associated with a higher infiltration of mouse macrophages within the tumors as assessed by F4/80 IHC labeling.Summary/Conclusion:The data presented here set the preclinical rationale and support a combination strategy of the novel CD47‐CD19 bispecific antibody TG‐1801 in B‐NHL with other B‐cell targeted mechanisms, including umbralisib and ublituximab.

262-LB: Human Pancreas-Derived Multipotent Stromal Cells Secrete Islet Regenerative Stimuli

We recently demonstrated that intra-pancreatic (iPan) delivery of conditioned media (CM) generated by human bone marrow-derived multipotent stromal cells (hBM-MSC) can act as a biotherapeutic to reduce hyperglycemia in STZ-treated mice by stimulating endogenous islet regeneration. We have established analogous stromal cell lines derived from human islet cultures that grow adherent to plastic and possess multipotent mesodermal differentiation in vitro. Furthermore, CM generated by these human pancreas-derived MSC (hPanc-MSC) contained proangiogenic stimuli within extracellular vesicles (EVs) that accelerated blood vessel regeneration after injection into ischemic hindlimbs. Herein, we investigated whether iPan-injection of CM generated by hPanc-MSC could stimulate islet regeneration in STZ-treated mice. We hypothesized that islet regenerative stimuli would be harboured within EVs secreted by hPanc-MSC. iPan-injection of unfractioned CM (6µg) generated by hBM-MSC and hPanc-MSC equally reduced hyperglycemia in STZ-treated mice, compared to mice injected with media control (*p<0.05). To determine whether islet regenerative stimuli was harboured within EVs, ultrafiltration was used to separate CM into EV enriched (EV+) or depleted (EV-) subfractions. Enrichment of EVs was validated by nanoscale flow cytometry and atomic force microscopy, and the EV+ and EV- hPANC-MSC subfractions were compared for protein content by label-free mass spectrometry. Expression of typical EV-associated proteins (CD9, CD81, CD63) were exclusive to the EV+ fraction and putative islet regenerative factors were detected in both the EV- and EV+ subfractions C. Mice iPan-injected with either EV+ or EV- CM sub-fractions showed significantly reduced hyperglycemia compared to unconditioned media controls (*p<0.05). These initial studies provide a foundation to further explore the use the hPanc-MSC secretome or related small-molecule pharmaceuticals to enhance islet-regeneration in situ. Disclosure T. Cooper: None. J. Ma: None. G.I. Bell: None. G. Lajoie: None. D.A. Hess: None.

The RANKL-RANK Axis: A Bone to Thymus Round Trip.

The identification of Receptor activator of nuclear factor kappa B ligand (RANKL) and its cognate receptor Receptor activator of nuclear factor kappa B (RANK) during a search for novel tumor necrosis factor receptor (TNFR) superfamily members has dramatically changed the scenario of bone biology by providing the functional and biochemical proof that RANKL signaling via RANK is the master factor for osteoclastogenesis. In parallel, two independent studies reported the identification of mouse RANKL on activated T cells and of a ligand for osteoprotegerin on a murine bone marrow-derived stromal cell line. After these seminal findings, accumulating data indicated RANKL and RANK not only as essential players for the development and activation of osteoclasts, but also for the correct differentiation of medullary thymic epithelial cells (mTECs) that act as mediators of the central tolerance process by which self-reactive T cells are eliminated while regulatory T cells are generated. In light of the RANKL-RANK multi-task function, an antibody targeting this pathway, denosumab, is now commonly used in the therapy of bone loss diseases including chronic inflammatory bone disorders and osteolytic bone metastases; furthermore, preclinical data support the therapeutic application of denosumab in the framework of a broader spectrum of tumors. Here, we discuss advances in cellular and molecular mechanisms elicited by RANKL-RANK pathway in the bone and thymus, and the extent to which its inhibition or augmentation can be translated in the clinical arena.

Histological Criteria that Distinguish Human and Mouse Bone Formed Within a Mouse Skeletal Repair Defect.

The effectiveness of autologous cell-based skeletal repair continues to be controversial in part because in vitro predictors of in vivo human bone formation by cultured human progenitor cells are not reliable. To assist in the development of in vivo assays of human osteoprogenitor potential, a fluorescence-based histology of nondecalcified mineralized tissue is presented that provides multiple criteria to distinguish human and host osteoblasts, osteocytes, and accumulated bone matrix in a mouse calvarial defect model. These include detection of an ubiquitously expressed red fluorescent protein reporter by the implanted human cells, antibodies specific to human bone sialoprotein and a human nuclear antigen, and expression of a bone/fibroblast restricted green fluorescent protein reporter in the host tissue. Using low passage bone marrow-derived stromal cells, robust human bone matrix formation was obtained. However, a striking feature is the lack of mouse bone marrow investment and osteoclasts within the human bone matrix. This deficiency may account for the accumulation of a disorganized human bone matrix that has not undergone extensive remodeling. These features, which would not be appreciated by traditional decalcified paraffin histology, indicate the human bone matrix is not undergoing active remodeling and thus the full differentiation potential of the implanted human cells within currently used mouse models is not being realized.

Correction to: Differential Expression of Cholinergic System Components in Human Induced Pluripotent Stem Cells, Bone Marrow-Derived Multipotent Stromal Cells, and Induced Pluripotent Stem Cell-Derived Multipotent Stromal Cells by Weist R, Flörkemeier T. Roger Y, Franke A, Schwanke K, Zweigerdt R, Martin U, Willbold E, and Hoffmann A. Stem Cells Dev 2017;27;3:166-183 DOI: 10.1089/scd.2017.0162.

Correction to: Differential Expression of Cholinergic System Components in Human Induced Pluripotent Stem Cells, Bone Marrow-Derived Multipotent Stromal Cells, and Induced Pluripotent Stem Cell-Derived Multipotent Stromal Cells by Weist R, Flörkemeier T. Roger Y, Franke A, Schwanke K, Zweigerdt R, Martin U, Willbold E, and Hoffmann A. Stem Cells Dev 2017;27;3:166-183 DOI: 10.1089/scd.2017.0162.