Primary Human Mesenchymal Stem Cells Research Articles

Gradient platform for combinatorial screening of thermoset polymers for biomedical applications

The goal of this work was to design a device for rapid screening of crosslinked thermoset polymers. This gradient curing platform is capable of yielding a library of polyesters with systematically varying mechanical and physicochemical properties and the resultant cellular response. A library of poly(xylitolsebacate) polyesters was prepared in this device by differential curing to yield a gradient polymer. The resultant polymer exhibits a gradient in the storage modulus (1 to 5 MPa), wettability (70° < water contact angle < 110°), degree of crosslinking, degradation rate (3–25% in 7 days), drug release and biological response (ability to support stem cell proliferation and differentiation) from one end of the polymer to the other. Primary human mesenchymal stem cells were cultured to assess the cellular response in vitro. Maximal stem cell proliferation and osteogenesis was observed on the highly crosslinked polyester segments that provide high stiffness, are hydrophobic and are slow degrading as compared to the lower cured counterparts. Under in vivo conditions, this material showed differential response across the gradient without displaying significant concerns for inflammation or infection. This gradient curing device is capable of ascertaining suitable curing conditions to obtain appropriate polymers for application specific requirements. This gradient platform was further used to identify optimal processing parameters to prepare three-dimensional tissue scaffolds such as electrospun fiber mats and porous foams. Thus, this versatile combinatorial platform is well suited for rapid screening of thermoset polymers for biomedical applications.

Efficient homology-directed gene editing by CRISPR/Cas9 in human stem and primary cells using tube electroporation

CRISPR/Cas9 efficiently generates gene knock-out via nonhomologous end joining (NHEJ), but the efficiency of precise homology-directed repair (HDR) is substantially lower, especially in the hard-to-transfect human stem cells and primary cells. Herein we report a tube electroporation method that can effectively transfect human stem cells and primary cells with minimal cytotoxicity. When applied to genome editing using CRISPR/Cas9 along with single stranded DNA oligonucleotide (ssODN) template in human induced pluripotent stem cells (iPSCs), up to 42.1% HDR rate was achieved, drastically higher than many reported before. We demonstrated that the high HDR efficiency can be utilized to increase the gene ablation rate in cells relevant to clinical applications, by knocking-out β2-microglobulin (B2M) in primary human mesenchymal stem cells (MSCs, 37.3% to 80.2%), and programmed death-1 (PD-1) in primary human T cells (42.6% to 58.6%). Given the generality and efficiency, we expect that the method will have immediate impacts in cell research as well as immuno- and transplantation therapies.

DNA topoisomerase II\u03b2 stimulates neurite outgrowth in neural differentiated human mesenchymal stem cells through regulation of Rho-GTPases (RhoA/Rock2 pathway) and Nurr1 expression

BackgroundDNA topoisomerase IIβ (topo IIβ) is known to regulate neural differentiation by inducing the neuronal genes responsible for critical neural differentiation events such as neurite outgrowth and axon guidance. However, the pathways of axon growth controlled by topo IIβ have not been clarified yet. Microarray results of our previous study have shown that topo IIβ silencing in neural differentiated primary human mesenchymal stem cells (hMSCs) significantly alters the expression pattern of genes involved in neural polarity, axonal growth, and guidance, including Rho-GTPases. This study aims to further analyze the regulatory role of topo IIβ on the process of axon growth via regulation of Rho-GTPases.Methods and resultsFor this purpose, topo IIβ was silenced in neurally differentiated hMSCs. Cells lost their morphology because of topo IIβ deficiency, becoming enlarged and flattened. Additionally, a reduction in both neural differentiation efficiency and neurite length, upregulation in RhoA and Rock2, downregulation in Cdc42 gene expression were detected. On the other hand, cells were transfected with topo IIβ gene to elucidate the possible neuroprotective effect of topo IIβ overexpression on neural-induced hMSCs. Topo IIβ overexpression prompted all the cells to exhibit neural cell morphology as characterized by longer neurites. RhoA and Rock2 expressions were downregulated, whereas Cdc42 expression was upregulated. Nurr1 expression level correlated with topo IIβ in both topo IIβ-overexpressed and -silenced cells. Furthermore, differential translocation of Rho-GTPases was detected by immunostaining in response to topo IIβ.ConclusionOur results suggest that topo IIβ deficiency could give rise to neurodegeneration through dysregulation of Rho-GTPases. However, further in-vivo research is needed to demonstrate if re-regulation of Rho GTPases by topo IIβ overexpression could be a neuroprotective treatment in the case of neurodegenerative diseases.

Magnesium phosphate ceramics incorporating a novel indene compound promote osteoblast differentiation in vitro and bone regeneration in vivo

Incorporating bioactive molecules into synthetic ceramic scaffolds is challenging. In this study, to enhance bone regeneration, a magnesium phosphate (MgP) ceramic scaffold was incorporated with a novel indene compound, KR-34893. KR-34893 induced the deposition of minerals and expression of osteoblast marker genes in primary human bone marrow mesenchymal stem cells (BMSCs) and a mouse osteoblastic MC3T3-E1 cell line. Analysis of the mode of action showed that KR-34893 induced the phosphorylation of MAPK/extracellular signal-regulated kinase and extracellular signal-regulated kinase, and subsequently the expression of bone morphogenetic protein 7, accompanied by SMAD1/5/8 phosphorylation. Accordingly, KR-34893 was incorporated into an MgP scaffold prepared by 3D printing at room temperature, followed by cement reaction. KR-34893-incorporated MgP (KR-MgP) induced the expression of osteoblast differentiation marker genes in vitro. In a rat calvaria defect model, KR-MgP scaffolds enhanced bone regeneration and increased bone volume compared with MgP scaffolds, as assessed by micro-computed tomography and histological analyses. In conclusion, we developed a method for producing osteoinductive MgP scaffolds incorporating a bioactive organic compound, without high temperature sintering. The KR-MgP scaffolds enhanced osteoblast activation in vitro and bone regeneration in vivo.

Nonmineralized and Mineralized Collagen Scaffolds Induce Differential Osteogenic Signaling Pathways in Human Mesenchymal Stem Cells.

The instructive capabilities of extracellular matrix components in progenitor cell differentiation have recently generated significant interest in the development of bioinspired materials for regenerative applications. Previously, a correlation was described between the osteogenic capabilities of nanoparticulate mineralized collagen glycosaminoglycan scaffolds (MC-GAG) and an autogenous activation of small mothers against decapentaplegic ( Smad1/5) in the canonical bone morphogenetic protein receptor (BMPR) pathway with a diminished extracellular signal regulated kinase 1/2 (ERK1/2) activation when compared to nonmineralized collagen glycosaminoglycan scaffolds (Col-GAG). This work utilizes a canonical BMPR inhibitor (dorsomorphin homologue 1, DMH1) and an inhibitor of the mitogen activated protein kinase/ERK kinase (MEK)/(ERK) cascade (PD98059) to characterize the necessity of each pathway for osteogenesis. While DMH1 inhibits runt-related transcription factor 2 (Runx2) and bone sialoprotein II (BSPII) gene expression of primary human mesenchymal stem cells (hMSCs) on MC-GAG, PD98059 inhibits BSPII expression on Col-GAG independent of Runx2 expression. DMH1 inhibits mineralization on both Col-GAG and MC-GAG, however, PD98059 only inhibits mineralization on Col-GAG. DMH1 inhibits both Smad1/5 phosphorylation and Runx2 protein expression, whereas PD98059 inhibits ERK1/2 and c-Jun amino-terminal kinase 1/2 (JNK1/2) phosphorylation without affecting Runx2. Thus, activation of the canonical BMPR signaling is necessary for osteogenic differentiation and mineralization of hMSCs on Col-GAG or MC-GAG. The MEK/ERK cascade, intimately tied to JNK activation, is necessary for Runx2-independent osteogenesis on Col-GAG, while completely dispensable in osteogenesis on MC-GAG.

Interleukin 1 β-induced SMAD2/3 linker modifications are TAK1 dependent and delay TGFβ signaling in primary human mesenchymal stem cells

BackgroundChondrogenic differentiation of mesenchymal stem cells (MSC) requires transforming growth factor beta (TGFβ) signaling. TGFβ binds to the type I receptor activin-like kinase (ALK)5 and results in C-terminal SMAD2/3 phosphorylation (pSMAD2/3C). In turn pSMAD2/3C translocates to the nucleus and regulates target gene expression. Inflammatory mediators are known to exert an inhibitory effect on MSC differentiation. In this study we investigated the effect of interleukin 1 β (IL1β) on SMAD2/3 signaling dynamics and post-translational modifications. ResultsCo-stimulation of MSC with TGFβ and IL1β did not affect peak pSMAD2C levels at 1h post-stimulation. Surprisingly, SMAD3 transcriptional activity, as determined by the CAGA12-luciferase reporter construct, was enhanced by co-stimulation of TGFβ and IL1β compared to TGFβ alone. Furthermore, IL1β stimulation induced CAGA12-luciferase activity in a SMAD dependent way. As SMAD function can be modulated independent of canonical TGFβ signaling through the SMAD linker domain, we studied SMAD2 linker phosphorylation at specific threonine and serine residues. SMAD2 linker threonine and serine modifications were observed within 1h following TGFβ, IL1β or TGFβ and IL1β stimulation. Upon co-stimulation linker modified SMAD2 accumulated in the cytoplasm and SMAD2/3 target gene transcription (ID1, JUNB) at 2–4h was inhibited. A detailed time course analysis of IL1β-induced SMAD2 linker modifications revealed a distinct temperospatial pattern compared to TGFβ. Co-stimulation with both factors resulted in a similar kinetic profile as TGFβ alone. Nevertheless, IL1β did subtly alter TGFβ-induced pSMAD2C levels between 8 and 24h post-stimulation, which was reflected by TGFβ target gene expression (PAI1, JUNB). Direct evidence for the importance of SMAD3 linker modifications for the effect of IL1β on TGFβ signaling was obtained by over-expression of SMAD3 or a SMAD3 linker phospho-mutant. Finally, an inhibitor screening was performed to identify kinases involved in SMAD2/3 linker modifications. We identified TAK1 kinase activity as crucial for IL1β-induced SMAD2 linker modifications and CAGA12-luciferase activity. ConclusionsTGFβ and IL1β signaling interact at the SMAD2/3 level in human primary MSC. Down-stream TGFβ target genes were repressed by IL1β independent of C-terminal SMAD2 phosphorylation. We demonstrate that SMAD2/3 linker modifications are required for this interplay and identified TAK1 as a crucial mediator of IL1β-induced TGFβ signal modulation.

Abstract: Differential Osteogenic Signaling Pathways in Human Mesenchymal Stem Cells on Collagen Glycosaminoglycan Scaffolds

Non-Mineralized and Mineralized Collagen Scaffolds Induce Differential Osteogenic Signaling Pathways in Human Mesenchymal Stem Cells INTRODUCTION: The instructive capabilities of extracellular matrix components in progenitor cell differentiation have recently generated significant interest in the development of bioinspired materials for regenerative applications. Previously, we described a correlation between the osteogenic capabilities of nanoparticulate mineralized collagen glycosaminoglycan scaffolds (MC-GAG) and an autogenous activation of Smad1/5 in the canonical bone morphogenetic protein receptor (BMPR) pathway with a diminished ERK1/2 activation when compared to non-mineralized scaffolds (Col-GAG). In this work, we utilize a canonical BMPR inhibitor, DMH1, and an inhibitor of the MEK/ERK cascade, PD98059, to characterize the necessity of each pathway for osteogenesis. METHODS: hMSCs were cultured in osteogenic media on Col-GAG or MC-GAG scaffolds. Scaffolds were untreated or treated with the DMH1 or PD98059 at 50 mM for 4 days, 14 days, 24 days, 4 weeks, and 8 weeks. Gene and protein expression were measured using quantitative RT-PCR and western blot analysis. Scaffolds were subjected to histochemical and micro-computed tomographic analyses. RESULTS: Inhibition of the BMPR signaling pathway inhibited Runx2 and BSPII gene expression of primary human mesenchymal stem cells (hMSCs) on MC-GAG. In contrast, inhibition of the MEK/ERK axis downregulated BSPII expression on Col-GAG independent of Runx2 expression. While inhibition of the BMPR signaling pathway resulted in decreased mineralization on both Col-GAG and MC-GAG, inhibition of the MEK/ERK axis only affected mineralization on Col-GAG. When the mechanistic detais were evaluated in greater detail, inhibition of the BMPR pathway reduced both Smad1/5 phosphorylation and Runx2 protein expression on both MC-GAG and Col-GAG. Inhibition of the MEK/ERK axis downregulated phosphorylation of ERK1/2 and JNK1/2 without affecting Smad1/5 phosphorylation or Runx2 expression. CONCLUSION: Interactions between hMSCs and collagen-based materials result in mechanistic differences in osteogenesis. Activation of the canonical BMPR signaling is required for osteogenic differentiation and mineralization of hMSCs on Col-GAG or MC-GAG. The MEK/ERK cascade, intimately tied to JNK activation, is necessary for Runx2-independent osteogenesis on Col-GAG, while completely dispensable in osteogenesis on MC-GAG.

Carvacrol promotes angiogenic paracrine potential and endothelial differentiation of human mesenchymal stem cells at low concentrations

ObjectivesPhenolic monoterpene compound, named Carvacrol, has been found to exert different biological outcomes. It has been accepted that the angiogenic activity of human mesenchymal stem cells was crucial in the pursuit of appropriate regeneration. In the current experiment, we investigated the contribution of Carvacrol on the angiogenic behavior of primary human mesenchymal stem cells. MethodsMesenchymal stem cells were exposed to Carvacrol in a dose ranging from 25 to 200μM for 48h. We measured cell survival rate by MTT assay and migration rate by a scratch test. The oxidative status was monitored by measuring SOD, GPx activity. The endothelial differentiation was studied by evaluating the level of VE-cadherin and vWF by real-time PCR and ELISA analyses. The content of VEGF and tubulogenesis behavior was monitored in vitro. We also conducted Matrigel plug in vivo CAM assay to assess the angiogenic potential of conditioned media from human mesenchymal stem cells after exposure to Carvacrol. ResultsCarvacrol was able to increase mesenchymal stem cell survival and migration rate (p<0.05). An increased activity of SOD was obtained while GPx activity unchanged or reduced. We confirmed the endothelial differentiation of stem cells by detecting vWF and VE-cadherin expression (p<0.05). The VEGF expression was increased and mesenchymal stem cells conditioned media improved angiogenesis tube formation in vitro (p<0.05). Moreover, histological analysis revealed an enhanced microvascular density at the site of Matrigel plug in CAM assay. ConclusionsOur data shed lights on the possibility of a Carvacrol to induce angiogenesis in human mesenchymal stem cells by modulating cell differentiation and paracrine angiogenic response.

A dual-application poly (dl-lactic-co-glycolic) acid (PLGA)-chitosan composite scaffold for potential use in bone tissue engineering

The development of patient-friendly alternatives to bone-graft procedures is the driving force for new frontiers in bone tissue engineering. Poly (dl-lactic-co-glycolic acid) (PLGA) and chitosan are well-studied and easy-to-process polymers from which scaffolds can be fabricated. In this study, a novel dual-application scaffold system was formulated from porous PLGA and protein-loaded PLGA/chitosan microspheres. Physicochemical and in vitro protein release attributes were established. The therapeutic relevance, cytocompatibility with primary human mesenchymal stem cells (hMSCs) and osteogenic properties were tested. There was a significant reduction in burst release from the composite PLGA/chitosan microspheres compared with PLGA alone. Scaffolds sintered from porous microspheres at 37 °C were significantly stronger than the PLGA control, with compressive strengths of 0.846 ± 0.272 MPa and 0.406 ± 0.265 MPa, respectively (p < 0.05). The formulation also sintered at 37 °C following injection through a needle, demonstrating its injectable potential. The scaffolds demonstrated cytocompatibility, with increased cell numbers observed over an 8-day study period. Von Kossa and immunostaining of the hMSC-scaffolds confirmed their osteogenic potential with the ability to sinter at 37 °C in situ.

Assessing cell-nanoparticle interactions by high content imaging of biocompatible iron oxide nanoparticles as potential contrast agents for magnetic resonance imaging

Stem cell tracking in cellular therapy and regenerative medicine is an urgent need, superparamagnetic iron oxide nanoparticles (IONPs) could be used as contrast agents in magnetic resonance imaging (MRI) that allows visualization of the implanted cells ensuring they reach the desired sites in vivo. Herein, we report the study of the interaction of 3,4-dihydroxyhydrocinnamic acid (DHCA) functionalized IONPs that have desirable properties for T2 - weighted MRI, with bone marrow-derived primary human mesenchymal stem cells (hMSCs). Using the multiparametric high-content imaging method, we evaluate cell viability, formation of reactive oxygen species, mitochondrial health, as well as cell morphology and determine that the hMSCs are minimally affected after labelling with IONPs. Their cellular uptake is visualized by transmission electron microscopy (TEM) and Prussian Blue staining, and quantified using an iron specific colourimetric method. In vitro and in vivo studies demonstrate that these IONPs are biocompatible and can produce significant contrast enhancement in T2-weighted MRI. Iron oxide nanoparticles are detected in vivo as hypointense regions in the liver up to two weeks post injection using 9.4 T MRI. These DHCA functionalized IONPs are promising contrast agents for stem cell tracking by T2-weighted MRI as they are biocompatible and show no evidence of cytotoxic effects on hMSCs.

A simple rocker-induced mechanical stimulus upregulates mineralization by human osteoprogenitor cells in fibrous scaffolds.

Biodegradable electrospun polycaprolactone scaffolds can be used to support bone‐forming cells and could fill a thin bony defect, such as in cleft palate. Oscillatory fluid flow has been shown to stimulate bone production in human progenitor cells in monolayer culture. The aim of this study was to examine whether bone matrix production by primary human mesenchymal stem cells from bone marrow or jaw periosteal tissue could be stimulated using oscillatory fluid flow supplied by a standard see‐saw rocker. This was investigated for cells in two‐dimensional culture and within electrospun polycaprolactone scaffolds. From day 4 of culture onwards, samples were rocked at 45 cycles/min for 1 h/day, 5 days/week (rocking group). Cell viability, calcium deposition, collagen production, alkaline phosphatase activity and vascular endothelial growth factor secretion were evaluated to assess the ability of the cells to undergo bone differentiation and induce vascularisation. Both cell types produced more mineralized tissue when subjected to rocking and supplemented with dexamethasone. Mesenchymal progenitors and primary human mesenchymal stem cells from bone marrow in three‐dimensional scaffolds upregulated mineral deposition after rocking culture as assessed by micro‐computed tomography and alizarin red staining. Interestingly, vascular endothelial growth factor secretion, which has previously been shown to be mechanically sensitive, was not altered by rocking in this system and was inhibited by dexamethasone. Rocker culture may be a cost effective, simple pretreatment for bone tissue engineering for small defects such as cleft palate.

Cationic osteogenic peptide P15-CSP coatings promote 3-D osteogenesis in poly(epsilon-caprolactone) scaffolds of distinct pore size.

P15-CSP is a biomimetic cationic fusion peptide that stimulates osteogenesis and inhibits bacterial biofilm formation when coated on 2-D surfaces. This study tested the hypothesis that P15-CSP coatings enhance 3-D osteogenesis in a porous but otherwise hydrophobic poly-(ɛ-caprolactone) (PCL) scaffold. Scaffolds of 84 µm and 141 µm average pore size were coated or not with Layer-by-Layer polyelectrolytes followed by P15-CSP, seeded with adult primary human mesenchymal stem cells (MSCs), and cultured 10 days in proliferation medium, then 21 days in osteogenic medium. Atomic analyses showed that P15-CSP was successfully captured by LbL. After 2 days of culture, MSCs adhered and spread more on P15-CSP coated pores than PCL-only. At day 10, all constructs contained nonmineralized tissue. At day 31, all constructs became enveloped in a "skin" of tissue that, like 2-D cultures, underwent sporadic mineralization in areas of high cell density that extended into some 141 µm edge pores. By quantitative histomorphometry, 2.5-fold more tissue and biomineral accumulated in edge pores versus inner pores. P15-CSP specifically promoted tissue-scaffold integration, fourfold higher overall biomineralization, and more mineral deposits in the outer 84 µm and inner 141 µm pores than PCL-only (p < 0.05). 3-D Micro-CT revealed asymmetric mineral deposition consistent with histological calcium staining. This study provides proof-of-concept that P15-CSP coatings are osteoconductive in PCL pore surfaces with 3-D topography. Biomineralization deeper than 150 µm from the scaffold edge was optimally attained with the larger 141 µm peptide-coated pores. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 2171-2181, 2017.

Mesenchymal stem cell detachment with trace trypsin is superior to EDTA for in vitro chemotaxis and adhesion assays

Trypsin is frequently used to dissociate mesenchymal stem cells (MSCs) for in vitro adhesion and chemotaxis assays. However, its potential impact on surface receptor degradation is poorly understood. The purpose of this study was to evaluate the effect of trypsin-EDTA exposure versus PBS-EDTA on MSC surface receptor integrity and function. Primary human MSCs were detached with PBS-EDTA alone, or Cell Dissociation Buffer followed by 30 s exposure to 0.05% w/v trypsin-EDTA (trace trypsin method, TT), or 0.25% w/v trypsin exposure for 2 or 5 min. Cells were characterized for surface integrity of β1 integrin (CD29) and PDGF Receptor (PDGF-R), and assessed in vitro for adhesion to atelocollagen-coated surfaces and migration to PDGF-BB. PBS-EDTA detachment fully preserved receptor integrity but routinely detached only half of the adherent cells and led to cell aggregates that failed to adhere evenly across the Transwell migration insert. Both CD29 and PDGF-R were significantly degraded by 0.25% trypsin detachment for 2 or 5 min compared to the TT method or PBS-EDTA (p < 0.05). Cells migrated optimally to PDGF-BB when detached with the TT method (3.1-fold vs α−MEM, p = 0.01). Cells attached optimally to atelocollagen when detached using the TT method or PBS-EDTA (6- to 10-fold vs 0.25% trypsin, p < 0.01). CDB followed by trace trypsin-EDTA exposure is recommended over PBS-EDTA to produce a single-cell MSC suspension that preserves receptor integrity and more reproducible receptor-mediated responses.

The cellular magnetic response and biocompatibility of biogenic zinc- and cobalt-doped magnetite nanoparticles

The magnetic moment and anisotropy of magnetite nanoparticles can be optimised by doping with transition metal cations, enabling their properties to be tuned for different biomedical applications. In this study, we assessed the suitability of bacterially synthesized zinc- and cobalt-doped magnetite nanoparticles for biomedical applications. To do this we measured cellular viability and activity in primary human bone marrow-derived mesenchymal stem cells and human osteosarcoma-derived cells. Using AC susceptibility we studied doping induced changes in the magnetic response of the nanoparticles both as stable aqueous suspensions and when associated with cells. Our findings show that the magnetic response of the particles was altered after cellular interaction with a reduction in their mobility. In particular, the strongest AC susceptibility signal measured in vitro was from cells containing high-moment zinc-doped particles, whilst no signal was observed in cells containing the high-anisotropy cobalt-doped particles. For both particle types we found that the moderate dopant levels required for optimum magnetic properties did not alter their cytotoxicity or affect osteogenic differentiation of the stem cells. Thus, despite the known cytotoxicity of cobalt and zinc ions, these results suggest that iron oxide nanoparticles can be doped to sufficiently tailor their magnetic properties without compromising cellular biocompatibility.

Isolation of Human Mesenchymal Stem Cells for Studying ErbB Receptor Signaling.

ErbB receptor signaling plays pivotal roles in tumorigenesis, cancer development, and drug resistance. A better understanding of ErbB signaling is required to achieve better clinical outcomes in cancer treatment. With increasing evidence showing the link between human mesenchymal stem cells (MSCs) and cancer, there is a growing interest in studying the role of ErbB receptor signaling in the regulation of MSCs. For this purpose, obtaining quality primary human MSCs is of great importance. This chapter describes the method of isolating primary human MSCs, aiming to offer researchers of this field a useful tool.

Theobromine Upregulates Osteogenesis by Human Mesenchymal Stem Cells In Vitro and Accelerates Bone Development in Rats.

Theobromine (THB) is one of the major xanthine-like alkaloids found in cacao plant and a variety of other foodstuffs such as tea leaves, guarana and cola nuts. Historically, THB and its derivatives have been utilized to treat cardiac and circulatory disorders, drug-induced nephrotoxicity, proteinuria and as an immune-modulator. Our previous work demonstrated that THB has the capacity to improve the formation of hydroxyl-apatite during tooth development, suggesting that it may also enhance skeletal development. With its excellent safety profile and resistance to pharmacokinetic elimination, we reasoned that it might be an excellent natural osteoanabolic supplement during pregnancy, lactation and early postnatal growth. To determine whether THB had an effect on human osteoprogenitors, we subjected primary human bone marrow mesenchymal stem cells (hMSCs) to osteogenic assays after exposure to THB in vitro and observed that THB exposure increased the rate of osteogenesis and mineralization by hMSCs. Moreover, THB exposure resulted in a list of upregulated mRNA transcripts that best matched an osteogenic tissue expression signature as compared to other tissue expression signatures archived in several databases. To determine whether oral administration of THB resulted in improved skeletal growth, we provided pregnant rats with chow supplemented with THB during pregnancy and lactation. After weaning, offspring received THB continuously until postnatal day 50 (approximately 10mgkg-1 day-1). Administration of THB resulted in neonates with larger bones, and 50-day-old offspring accumulated greater body mass, longer and thicker femora and superior tibial trabecular parameters. The accelerated growth did not adversely affect the strength and resilience of the bones. These results indicate that THB increases the osteogenic potential of bone marrow osteoprogenitors, and dietary supplementation of a safe dose of THB to expectant mothers and during the postnatal period could accelerate skeletal development in their offspring.

Combined effect of grain refinement and surface modification of pure titanium on the attachment of mesenchymal stem cells and osteoblast-like SaOS-2 cells

Surface modification is an important step in production of medical implants. Surface roughening creates additional surface area to enhance the bonding between the implant and the bone. Recent research provided a means to alter the microstructure of titanium by severe plastic deformation (SPD) in order to increase its strength, and thereby reduce the size of the implants (specifically, their diameter). The purpose of the present study was to examine the effect of bulk microstructure of commercially pure titanium with coarse-grained (CG) and ultrafine-grained (UFG) bulk structure on the surface state of these materials after surface modification by sand blasting and acid etching (SLA). It was shown that SLA-modified surface characteristics, in particular, roughness, chemistry, and wettability, were affected by prior SPD processing.Additionally, biocompatibility of UFG titanium was examined using osteosarcoma cell line SaOS-2 and primary human adipose-derived mesenchymal stem cell (adMSC) cultures. Enhanced cell viability as well as increased matrix mineralization during osteogenic differentiation of MSCs on the surface of ultrafine-grained titanium was shown.

Chemotherapy-induced Dkk-1 expression by primary human mesenchymal stem cells is p53 dependent.

Mesenchymal stem cells (MSCs) are abundant throughout the body and regulate signaling within tumor microenvironments. Wnt signaling is an extrinsically regulated pathway that has been shown to regulate tumorigenesis in many types of cancer. After evaluating a panel of Wnt activating and inhibiting molecules, we show that primary human MSCs increase the expression of Dkk-1, an inhibitor of Wnt signaling, into the extracellular environment following chemotherapy exposure in a p53-dependent manner. Dkk-1 has been shown to promote tumor growth in several models of malignancy, suggesting that MSC-derived Dkk-1 could counteract the intent of cytotoxic chemotherapy, and that pharmacologic inhibition of Dkk-1 in patients receiving chemotherapy treatment for certain malignancies may be warranted.

Abstract B11: Targeting mesenchymal cells in the tumor stroma by GPX4 inhibition

Abstract Fibroblast-like mesenchymal cells are the most abundant component of tumor stroma and have been shown to promote tumor progression. However, there are no therapeutic agents that target these cells. Developing therapeutics against mesenchymal cells has been challenging because they lack clear “druggable” targets. Here we used a phenotypic screening approach to identify therapeutic targets in bone-marrow mesenchymal stem cells (MSCs), precursors of cancer-associated fibroblasts, that are reported to induce a metastatic phenotype in breast cancer cells (Karnoub et al, Nature 2007). Using the in vitro phenotype of enhanced migration of breast cancer cells (GFP-labeled MDA-MB-231; MDA) induced by MSCs (3-fold faster than MDA alone), we identified inflammatory signaling and glutathione peroxidase 4 as potential targets in MSCs. To validate this screening model, we first confirmed that MSCs can enhance metastasis using orthotopic xenografts of luciferase-labeled MDA cells co-injected with primary human MSCs in NOD-SCID mice. Mice with MSC+MDA tumors had 5-fold greater thoracic bioluminescence (corresponding to lung metastasis) than mice with MDA tumors alone. We then performed gene-expression profiling of MSC+MDA co-cultures in vitro compared to cells grown alone. The top pathway upregulated in co-cultures was the interferon pathway, suggesting that an inflammatory response follows MSC-MDA interactions. Using publicly available gene-expression datasets, we prioritized transcripts that are expressed in patient stroma and correlate with poor survival in a meta-analysis of 20 whole-tumor datasets. To determine if these transcripts are necessary for MSC-induced metastatic behavior, we performed shRNA knockdown and measured effects on in vitro migration of MSC+MDA co-cultures compared to normal endothelial cells. Knockdown of 9 genes was specific for MSC+MDA migration but the effects were weak. This suggested functional redundancy and indicated that targeting individual upregulated genes is insufficient to block the phenotype. We then performed a small-molecule screen on the MSC+MDA migration phenotype, counter-screening hits on endothelial cell migration. Only one compound, RSL3, showed a large and selective inhibition of MSC+MDA migration. RSL3 was selectively toxic to MSCs with no effect on MDA cells. The target of RSL3 was recently identified to be glutathione peroxidase 4 (GPX4) that metabolizes lipid peroxides. To identify lipid mediators of RSL3 toxicity, we profiled lipid levels in RSL3-treated MSC+MDA co-cultures and found that the top changes were lipoxygenase products of arachidonic acid. Pre-treatment with zileuton or PD146176, that inhibit 5- and 15-lipoxygenase respectively, abrogated RSL3's toxic effects on MSCs. We found that MSCs contain &amp;gt;10-fold higher lipoxygenase products than MDA cells, highlighting an inflammatory role of MSCs. Fibroblasts from different tissues (lungs, spleen, breast, skin) were similarly sensitive to RSL3, identifying a previously unrecognized vulnerability in these mesenchymal cells. Since RSL3 is a tool compound with poor plasma stability, we used GPX4 shRNA knockdown (KD) to determine in vivo effects of depleting MSCs in MSC+MDA xenografts. Unexpectedly, we found accelerated tumor growth in GPX4 KD tumors compared with controls. However, this is consistent with previous reports showing that mesenchymal cells restrain tumor growth and can have both pro- and anti-tumor effects. Histological examination revealed increased myeloid cell infiltration into GPX4 KD tumors reflecting increased inflammation mediated by lipoxygenase products like leukotriene B4. In summary, phenotypic screening has identified GPX4 inhibition as a novel approach to target mesenchymal cells. This approach may be particularly effective at recruiting immune/ inflammatory cells into tumors with the exciting possibility of synergizing with cancer immunotherapy. Citation Format: Shrikanta Chattopadhyay, Cherrie Huang, Ninib Baryawno, Nicolas Severe, Vasanthi Viswanathan, Zarko Boskovic, Siddhartha Mukherjee, Jeff Gentry, Ben Wittner, Sridhar Ramaswamy, Alykhan Shamji, David Scadden, Stuart Schreiber. Targeting mesenchymal cells in the tumor stroma by GPX4 inhibition. [abstract]. In: Proceedings of the AACR Special Conference: Function of Tumor Microenvironment in Cancer Progression; 2016 Jan 7–10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2016;76(15 Suppl):Abstract nr B11.

Nanoparticulate Mineralized Collagen Scaffolds and BMP-9 Induce a Long-Term Bone Cartilage Construct in Human Mesenchymal Stem Cells.

Engineering the osteochondral junction requires fabrication of a microenvironment that supports both osteogenesis and chondrogenesis. Multiphasic scaffold strategies utilizing a combination of soluble factors and extracellular matrix components are ideally suited for such applications. In this work, the contribution of an osteogenic nanoparticulate mineralized glycosaminoglycan scaffold (MC-GAG) and a dually chondrogenic and osteogenic growth factor, BMP-9, in the differentiation of primary human mesenchymal stem cells (hMSCs) is evaluated. Although 2D cultures demonstrate alkaline phosphatase activity and mineralization of hMSCs induced by BMP-9, MC-GAG scaffolds do not demonstrate significant differences in the collagen I expression, osteopontin expression, or mineralization. Instead, BMP-9 increases expression of collagen II, Sox9, aggrecan (ACAN), and cartilage oligomeric protein. However, the hypertrophic chondrocyte marker, collagen X, is not elevated with BMP-9 treatment. In addition, histologic analyses demonstrate that while BMP-9 does not increase mineralization, BMP-9 treatment results in an increase of sulfated glycosaminoglycans. Thus, the combination of BMP-9 and MC-GAG stimulates chondrocytic and osteogenic differentiation of hMSCs.