Aberrant Expression of COX-2 and FOXG1 in Infrapatellar Fat Pad-Derived ASCs from Pre-Diabetic Donors.

Osteoarthritis (OA) is a degenerative joint disease resulting in limited mobility and severe disability. Type II diabetes mellitus (T2D) is a weight-independent risk factor for OA, but a link between the two diseases has not been elucidated. Adipose stem cells (ASCs) isolated from the infrapatellar fat pad (IPFP) may be a viable regenerative cell for OA treatment. This study analyzed the expression profiles of inflammatory and adipokine-related genes in IPFP-ASCs of non-diabetic (Non-T2D), pre-diabetic (Pre-T2D), and T2D donors. Pre-T2D ASCs exhibited a substantial decrease in levels of mesenchymal markers CD90 and CD105 with no change in adipogenic differentiation compared to Non-T2D and T2D IPFP-ASCs. In addition, Cyclooxygenase-2 (COX-2), Forkhead box G1 (FOXG1) expression and prostaglandin E2 (PGE2) secretion were significantly increased in Pre-T2D IPFP-ASCs upon stimulation by interleukin-1 beta (IL-1ß). Interestingly, M1 macrophages exhibited a significant reduction in expression of pro-inflammatory markers TNFα and IL-6 when co-cultured with Pre-T2D IPFP-ASCs. These data suggest that the heightened systemic inflammation associated with untreated T2D may prime the IPFP-ASCs to exhibit enhanced anti-inflammatory characteristics via suppressing the IL-6/COX-2 signaling pathway. In addition, the elevated production of PGE2 by the Pre-T2D IPFP-ASCs may also suggest the contribution of pre-diabetic conditions to the onset and progression of OA.

Human Mesenchymal Stem Cell-Derived Miniature Joint System for Disease Modeling and Drug Testing.

Diseases of the knee joint such as osteoarthritis (OA) affect all joint elements. An in vitro human cell-derived microphysiological system capable of simulating intraarticular tissue crosstalk is desirable for studying etiologies/pathogenesis of joint diseases and testing potential therapeutics. Herein, a human mesenchymal stem cell-derived miniature joint system (miniJoint) is generated, in which engineered osteochondral complex, synovial-like fibrous tissue, and adipose tissue are integrated into a microfluidics-enabled bioreactor. This novel design facilitates different tissues communicating while still maintaining their respective phenotypes. The miniJoint exhibits physiologically relevant changes when exposed to interleukin-1ß mediated inflammation, which are similar to observations in joint diseases in humans. The potential of the miniJoint in predicting in vivo efficacy of drug treatment is confirmed by testing the "therapeutic effect" of the nonsteroidal anti-inflammatory drug, naproxen, as well as four other potential disease-modifying OA drugs. The data demonstrate that the miniJoint recapitulates complex tissue interactions, thus providing a robust organ chip model for the study of joint pathology and the development of novel therapeutic interventions.

Adipose Tissue-Derived Stem Cells Retain Their Adipocyte Differentiation Potential in Three-Dimensional Hydrogels and Bioreactors †.

Osteoarthritis (OA) is a common joint disorder with a significant economic and healthcare impact. The knee joint is composed of cartilage and the adjoining bone, a synovial capsule, the infrapatellar fat pad (IPFP), and other connective tissues such as tendons and ligaments. Adipose tissue has recently been highlighted as a major contributor to OA through strong inflammation mediating effects. In this study, methacrylated gelatin (GelMA) constructs seeded with adipose tissue-derived mesenchymal stem cells (ASCs) and cultured in a 3D printed bioreactor were investigated for use in microphysiological systems to model adipose tissue in the knee joint. Four patient-derived ASC populations were seeded at a density of 20 million cells/mL in GelMA. Live/Dead and boron-dipyrromethene/4',6-diamidino-2-phenylindole (BODIPY/DAPI) staining of cells within the constructs demonstrated robust cell viability after 28 days in a growth (control) medium, and robust cell viability and lipid accumulation in adipogenic differentiation medium. qPCR gene expression analysis and protein analysis demonstrated an upregulated expression of key adipogenesis-associated genes. Overall, these data indicate that ASCs retain their adipogenic potential when seeded within GelMA hydrogels and cultured within perfusion bioreactors, and thus can be used in a 3D organ-on-a-chip system to study the role of the IPFP in the pathobiology of the knee OA.

Interleukin-4 overexpressing mesenchymal stem cells within gelatin-based microribbon hydrogels enhance bone healing in a murine long bone critical-size defect model.

Mesenchymal stem cell (MSC)-based therapy is a promising strategy for bone repair. Furthermore, the innate immune system, and specifically macrophages, plays a crucial role in the differentiation and activation of MSCs. The anti-inflammatory cytokine Interleukin-4 (IL-4) converts pro-inflammatory M1 macrophages into a tissue regenerative M2 phenotype, which enhances MSC differentiation and function. We developed lentivirus-transduced IL-4 overexpressing MSCs (IL-4 MSCs) that continuously produce IL-4 and polarize macrophages toward an M2 phenotype. In the current study, we investigated the potential of IL-4 MSCs delivered using a macroporous gelatin-based microribbon (µRB) scaffold for healing of critical-size long bone defects in Mice. IL-4 MSCs within µRBs enhanced M2 marker expression without inhibiting M1 marker expression in the early phase, and increased macrophage migration into the scaffold. Six weeks after establishing the bone defect, IL-4 MSCs within µRBs enhanced bone formation and helped bridge the long bone defect. IL-4 MSCs delivered using macroporous µRB scaffold is potentially a valuable strategy for the treatment of critical-size long bone defects.

Macrophage Effects on Mesenchymal Stem Cell Osteogenesis in a Three-Dimensional In Vitro Bone Model.

As musculoskeletal (MSK) disorders continue to increase globally, there is an increased need for novel, in vitro models to efficiently study human bone physiology in the context of both healthy and diseased conditions. For these models, the inclusion of innate immune cells is critical. Specifically, signaling factors generated from macrophages play key roles in the pathogenesis of many MSK processes and diseases, including fracture, osteoarthritis, infection etc. In this study, we aim to engineer three-dimensional (3D) and macrophage-encapsulated bone tissues in vitro, to model cell behavior, signaling, and other biological activities in vivo, in comparison to current two-dimensional models. We first investigated and optimized 3D culture conditions for macrophages, and then co-cultured macrophages with mesenchymal stem cells (MSCs), which were induced to undergo osteogenic differentiation to examine the effect of macrophage on new bone formation. Seeded within a 3D hydrogel scaffold fabricated from photocrosslinked methacrylated gelatin, macrophages maintained high viability and were polarized toward an M1 or M2 phenotype. In co-cultures of macrophages and human MSCs, MSCs displayed immunomodulatory activities by suppressing M1 and enhancing M2 macrophage phenotypes. Lastly, addition of macrophages, regardless of polarization state, increased MSC osteogenic differentiation, compared with MSCs alone, with proinflammatory M1 macrophages enhancing new bone formation most effectively. In summary, this study illustrates the important roles that macrophage signaling and inflammation play in bone tissue formation.

Precise immunomodulation of the M1 to M2 macrophage transition enhances mesenchymal stem cell osteogenesis and differs by sex.

OBJECTIVES: Up to 10% of fractures result in undesirable outcomes, for which female sex is a risk factor. Cellular sex differences have been implicated in these different healing processes. Better understanding of the mechanisms underlying bone healing and sex differences in this process is key to improved clinical outcomes. This study utilized a macrophage-mesenchymal stem cell (MSC) coculture system to determine: 1) the precise timing of proinflammatory (M1) to anti-inflammatory (M2) macrophage transition for optimal bone formation; and 2) how such immunomodulation was affected by male versus female cocultures. METHODS: A primary murine macrophage-MSC coculture system was used to demonstrate the optimal transition time from M1 to M2 (polarized from M1 with interleukin (IL)-4) macrophages to maximize matrix mineralization in male and female MSCs. Outcome variables included Alizarin Red staining, alkaline phosphatase (ALP) activity, and osteocalcin protein secretion. RESULTS: We found that 96 hours of M1 phenotype in male cocultures allowed for maximum matrix mineralization versus 72 hours in female cocultures. ALP activity and osteocalcin secretion were also enhanced with the addition of IL-4 later in male versus female groups. The sex of the cells had a statistically significant effect on the optimal IL-4 addition time to maximize osteogenesis. CONCLUSION: These results suggest that: 1) a 72- to 96-hour proinflammatory environment is critical for optimal matrix mineralization; and 2) there are immunological differences in this coculture environment due to sex. Optimizing immunomodulation during fracture healing may enhance and expedite the bone regeneration response. These findings provide insight into precise immunomodulation for enhanced bone healing that is sex-specific.Cite this article: K. Nathan, L. Y. Lu, T. Lin, J. Pajarinen, E. Jämsen, J-F. Huang, M. Romero-Lopez, M. Maruyama, Y. Kohno, Z. Yao, S. B. Goodman. Precise immunomodulation of the M1 to M2 macrophage transition enhances mesenchymal stem cell osteogenesis and differs by sex. Bone Joint Res 2019;8:481-488. DOI: 10.1302/2046-3758.810.BJR-2018-0231.R2.

Optimization and Characterization of Calcium Phosphate Transfection in Mesenchymal Stem Cells.

IMPACT STATEMENT: Mesenchymal stem cells (MSCs) are a promising tool for cell therapy, and gene-modified MSCs further expand their applications. To take full advantage of MSCs as a therapeutic approach, developing effective gene transfer methods is critical. Calcium phosphate transfection is well-established and safe, but the protocols need to be optimized according to different cell types. Currently, there is no optimized protocol for MSCs. This study optimized the protocol of calcium phosphate transfection for MSCs and highlighted the importance of serum during the process of transfection. More interestingly, the behavior of gene overexpression in MSCs in the in vivo environment was verified.

Osteogenic ability of rat bone marrow concentrate is at least as efficacious as mesenchymal stem cells in vitro.

Cell therapy using bone marrow concentrate (BMC) or purified and expanded mesenchymal stem cells (MSCs) has been shown to have a promising osteogenic capacity. However, few studies have directly compared their relative osteogenic ability. The aim of this study was to compare the osteogenic ability of BMC isolated by density gradient centrifugation with bone marrow-derived MSCs in vitro using the cells of 3-month-old Sprague-Dawley rats. The isolated cells were seeded onto 24-well plates (1 × 105 cells/well) and cultured in control growth media, osteogenic media with dexamethasone, or media without dexamethasone (which simulated the in vivo tissue environment). Alkaline phosphatase activity at week 2, osteocalcin using quantitative real-time polymerase chain reaction at week 4, and Alizarin red staining at week 4 were evaluated. In the osteogenic media with dexamethasone, BMC showed equivalent (osteocalcin) or even greater (Alizarin red staining) osteogenic ability compared to MSCs, suggesting that cross-talk among various cells in the BMC leads to greater osteogenesis. Furthermore, in the osteogenic media without dexamethasone, BMC showed equivalent (osteocalcin) or a trend for greater (Alizarin red staining) bone formation than MSCs alone. Our results suggest that BMC has at least comparable bone regeneration potential to MSCs. © 2019 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater 107B:2500-2506, 2019.

Preconditioned or IL4-Secreting Mesenchymal Stem Cells Enhanced Osteogenesis at Different Stages.

IMPACT STATEMENT: Pathogen-associated molecular patterns, damage-associated molecular patterns, and other noxious stimuli activate macrophages to induce the proinflammatory responses. Modulation of inflammatory macrophages (M1) into an anti-inflammatory tissue repair macrophage (M2) phenotype at the appropriate time optimizes bone remodeling and regeneration. Simulating the proinflammatory stimuli by using preconditioned mesenchymal stem cells (MSCs) at an earlier stage, and alleviate the inflammation by using IL4-secreting MSCs at a later stage could further optimize bone regeneration in chronic inflammatory conditions, including periprosthetic osteolysis.

Treating Titanium Particle-Induced Inflammation with Genetically Modified NF-κB Sensing IL-4 Secreting or Preconditioned Mesenchymal Stem Cells in Vitro.

Titanium and titanium-based alloys are widely used in orthopaedic implants. Total joint replacement is very successful; however, the foreign body response and chronic inflammation caused by implant-derived biomaterial debris still remain as unsolved issues. Aseptic loosening accompanied by wear debris-induced osteolysis (bone loss) is one of the most frequent causes for late failure and revision surgery. Mesenchymal stem cells (MSCs) and IL-4 may be possible treatment strategies because of their immunomodulatory properties. We investigated the efficacy of novel MSC-based treatments on immunomodulation and osteogenic differentiation in an innovative cell coculture model of titanium particle-induced inflammation in the periprosthetic tissues. MSCs and macrophages were collected from the bone marrow of Balb/c mice. Both MSCs and macrophages (representing endogenous cells at the periprosthetic tissue) were seeded on the bottom wells of the 24-well transwell plates. We generated genetically modified NF-κB sensing IL-4 secreting MSCs (inflammatory responsive MSCs) and MSCs preconditioned by lipopolysaccharide and TNF-α to further enhance their immunomodulatory function. These modified MSCs (representing exogenous therapeutic cells implanted to the periprosthetic tissue) were seeded on the upper chambers of the transwell plates. These cocultures were then exposed to titanium particles for 7 days. NF-κB sensing IL-4 secreting MSCs showed strong immunomodulation (significantly reduced TNF-α and induced Arg1 expression) and promoted early osteogenesis (significantly induced Runx2, ALP, and ß-catenin as well as reduced Smurf2 expression) at day 7. IL-4 secreting MSCs also decreased TNF-α protein secretion as early as day 3 and increased IL-1ra protein secretion at day 7, suggesting efficacious immunomodulation of particle-induced inflammation. Preconditioned MSCs did not show significant immunomodulation in this short-term experiment, but ALP and ß-catenin expression were significantly induced at day 7. Our results suggest that genetically modified IL-4 secreting MSCs and preconditioned MSCs have the potential to optimize bone regeneration in inflammatory conditions including periprosthetic osteolysis.

Trained murine mesenchymal stem cells have anti-inflammatory effect on macrophages, but defective regulation on T-cell proliferation.

Mesenchymal stem cell (MSC)-mediated immunomodulation affects both innate and adaptive immune systems. These responses to environmental cues, such as pathogen-associated molecular patterns, damage-associated molecular patterns, or proinflammatory cytokines, are crucial for resolution of inflammation, as well as successful tissue healing and regeneration. We observed that intermittent, repeated exposure of MSCs to LPS induced stronger NF-κB activation than singular stimulation. A similar phenomenon, named innate immune memory or trained immunity, has been reported with macrophages. However, the potential regulation of "immune memory" in nonclassic immune cells, such as MSCs, has not been reported. In the current study, we chose IFN-γ plus TNF-α restimulation-induced iNOS expression as a model of MSC activation, because IFN-γ and TNF-α play crucial roles in MSC-mediated immunomodulation. The iNOS expression was enhanced in LPS-trained MSCs, 3 d after a washout period following primary stimulation. LPS-trained MSCs enhanced the anti-inflammatory (arginase 1 and CD206) marker expression, but decreased the proinflammatory marker (TNF-α, IL-1ß, iNOS, and IL-6) expression using an MSC-macrophage coculture model. In contrast, LPS-trained MSCs demonstrated a defective regulation on CD4 T-cell proliferation. Mechanistic studies suggested that histone methylation and the JNK pathway are involved in LPS-trained immunomodulation in MSCs. Our results demonstrate differential immunomodulatory effects of trained MSCs on macrophages and T cells. These immunomodulatory consequences are critical, because they will have a major impact on current MSC-based cell therapies.-Lin, T., Pajarinen, J., Kohno, Y., Huang, J.-F., Maruyama, M., Romero-Lopez, M., Nathan, K., Yao, Z., Goodman, S. B. Trained murine mesenchymal stem cells have anti-inflammatory effect on macrophages, but defective regulation on T-cell proliferation.

NFκB sensing IL-4 secreting mesenchymal stem cells mitigate the proinflammatory response of macrophages exposed to polyethylene wear particles.

Total joint replacement is a highly effective treatment for patients with end-stage arthritis. Proinflammatory macrophages (M1) mediate wear particle-associated inflammation and bone loss. Anti-inflammatory macrophages (M2) help resolve tissue damage and favor bone regeneration. Mesenchymal stem cell (MSC)-based therapy mitigates the M1 dominated inflammatory reaction and favorably modulates the bone remodeling process. In the current study, the immunomodulating ability of (1) unmodified MSCs, (2) MSCs preconditioned by NFκB stimulating ligands [lipopolysaccharide (LPS) plus TNFα], and (3) genetically modified MSCs that secrete IL-4 as a response to NFκB activation (NFκB-IL4) was compared in a macrophage/MSC co-culture system. Sterile or LPS-contaminated ultra-high molecular weight polyethylene particles were used to induce the proinflammatory responses in the macrophages. Contaminated particles induced M1 marker expression (TNFα, IL1ß, and iNOS), while NFκB-IL4 MSCs modulated the macrophages from an M1 phenotype into a more favorable M2 phenotype (Arginase 1/Arg 1 and CD206 high). The IL4 secretion by NFκB-IL4 MSCs was significantly induced by the contaminated particles. The induction of Arg 1 and CD206 in macrophages via the preconditioned or naïve MSCs was negligible when compared with NFκB-IL4 MSC. Our findings indicated that NFκB-IL4 MSCs have the "on-demand" immunomodulatory ability to mitigate wear particle-associated inflammation with minimal adverse effects. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 106A: 2744-2752, 2018.

Transplanted interleukin-4--secreting mesenchymal stromal cells show extended survival and increased bone mineral density in the murine femur.

BACKGROUND: Mesenchymal stromal cell (MSC)-based therapy has great potential to modulate chronic inflammation and enhance tissue regeneration. Crosstalk between MSC-lineage cells and polarized macrophages is critical for bone formation and remodeling in inflammatory bone diseases. However, the translational application of this interaction is limited by the short-term viability of MSCs after cell transplantation. METHODS: Three types of genetically modified (GM) MSCs were created: (1) luciferase-expressing reporter MSCs; (2) MSCs that secrete interleukin (IL)-4 either constitutively; and (3) MSCs that secrete IL-4 as a response to nuclear factor kappa-light-chain-enhancer of activated B cell (NFκB) activation. Cells were injected into the murine distal femoral bone marrow cavity. MSC viability and bone formation were examined in vivo. Cytokine secretion was determined in a femoral explant organ culture model. RESULTS: The reporter MSCs survived up to 4 weeks post-implantation. No difference in the number of viable cells was found between high (2.5 × 106) and low (0.5 × 106) cell-injected groups. Injection of 2.5 × 106 reporter MSCs increased local bone mineral density at 4 weeks post-implantation. Injection of 0.5 × 106 constitutive IL-4 or NFκB-sensing IL-4-secreting MSCs increased bone mineral density at 2 weeks post-implantation. In the femoral explant organ culture model, LPS treatment induced IL-4 secretion in the NFκB-sensing IL-4-secreting MSC group and IL-10 secretion in all the femur samples. No significant differences in tumor necrosis factor (TNF)α and IL-1ß secretion were observed between the MSC-transplanted and control groups in the explant culture. DISCUSSION: Transplanted GM MSCs demonstrated prolonged cell viability when transplanted to a compatible niche within the bone marrow cavity. GM IL-4-secreting MSCs may have great potential to enhance bone regeneration in disorders associated with chronic inflammation.

3D Mathematical Modeling of Glioblastoma Suggests That Transdifferentiated Vascular Endothelial Cells Mediate Resistance to Current Standard-of-Care Therapy.

Glioblastoma (GBM), the most aggressive brain tumor in human patients, is decidedly heterogeneous and highly vascularized. Glioma stem/initiating cells (GSC) are found to play a crucial role by increasing cancer aggressiveness and promoting resistance to therapy. Recently, cross-talk between GSC and vascular endothelial cells has been shown to significantly promote GSC self-renewal and tumor progression. Furthermore, GSC also transdifferentiate into bona fide vascular endothelial cells (GEC), which inherit mutations present in GSC and are resistant to traditional antiangiogenic therapies. Here we use three-dimensional mathematical modeling to investigate GBM progression and response to therapy. The model predicted that GSCs drive invasive fingering and that GEC spontaneously form a network within the hypoxic core, consistent with published experimental findings. Standard-of-care treatments using DNA-targeted therapy (radiation/chemo) together with antiangiogenic therapies reduced GBM tumor size but increased invasiveness. Anti-GEC treatments blocked the GEC support of GSCs and reduced tumor size but led to increased invasiveness. Anti-GSC therapies that promote differentiation or disturb the stem cell niche effectively reduced tumor invasiveness and size, but were ultimately limited in reducing tumor size because GECs maintain GSCs. Our study suggests that a combinatorial regimen targeting the vasculature, GSCs, and GECs, using drugs already approved by the FDA, can reduce both tumor size and invasiveness and could lead to tumor eradication. Cancer Res; 77(15); 4171-84. ©2017 AACR.

Recapitulating the human tumor microenvironment: Colon tumor-derived extracellular matrix promotes angiogenesis and tumor cell growth.

Extracellular matrix (ECM) is an essential and dynamic component of all tissues and directly affects cellular behavior by providing both mechanical and biochemical signaling cues. Changes in ECM can alter tissue homeostasis, potentially leading to promotion of cellular transformation and the generation of tumors. Therefore, understanding ECM compositional changes during cancer progression is vital to the development of targeted treatments. Previous efforts to reproduce the native 3D cellular microenvironment have utilized protein gels and scaffolds that incompletely recapitulate the complexity of native tissues. Here, we address this problem by extracting and comparing ECM from normal human colon and colon tumor that had metastasized to liver. We found differences in protein composition and stiffness, and observed significant differences in vascular network formation and tumor growth in each of the reconstituted matrices, both in vitro and in vivo. We studied free/bound ratios of NADH in the tumor and endothelial cells using Fluorescence Lifetime Imaging Microscopy as a surrogate for the metabolic state of the cells. We observed that cells seeded in tumor ECM had higher relative levels of free NADH, consistent with a higher glycolytic rate, than those seeded in normal ECM. These results demonstrate that the ECM plays an important role in the growth of cancer cells and their associated vasculature.

Multiscale Modeling of Glioblastoma Suggests that the Partial Disruption of Vessel/Cancer Stem Cell Crosstalk Can Promote Tumor Regression Without Increasing Invasiveness.

OBJECTIVE: In glioblastoma, the crosstalk between vascular endothelial cells (VECs) and glioma stem cells (GSCs) has been shown to enhance tumor growth. We propose a multiscale mathematical model to study this mechanism, explore tumor growth under various initial and microenvironmental conditions, and investigate the effects of blocking this crosstalk. METHODS: We develop a hybrid continuum-discrete model of highly organized vascularized tumors. VEC-GSC crosstalk is modeled via vascular endothelial growth factor (VEGF) production by tumor cells and by secretion of soluble factors by VECs that promote GSC self-renewal and proliferation. RESULTS: VEC-GSC crosstalk increases both tumor size and GSC fraction by enhancing GSC activity and neovascular development. VEGF promotes vessel formation, and larger VEGF sources typically increase vessel numbers, which enhances tumor growth and stabilizes the tumor shape. Increasing the initial GSC fraction has a similar effect. Partially disrupting the crosstalk by blocking VEC secretion of GSC promoters reduces tumor size but does not increase invasiveness, which is in contrast to antiangiogenic therapies, which reduce tumor size but may significantly increase tumor invasiveness. SIGNIFICANCE: Multiscale modeling supports the targeting of VEC-GSC crosstalk as a promising approach for cancer therapy.

3D microtumors in vitro supported by perfused vascular networks.

There is a growing interest in developing microphysiological systems that can be used to model both normal and pathological human organs in vitro. This "organs-on-chips" approach aims to capture key structural and physiological characteristics of the target tissue. Here we describe in vitro vascularized microtumors (VMTs). This "tumor-on-a-chip" platform incorporates human tumor and stromal cells that grow in a 3D extracellular matrix and that depend for survival on nutrient delivery through living, perfused microvessels. Both colorectal and breast cancer cells grow vigorously in the platform and respond to standard-of-care therapies, showing reduced growth and/or regression. Vascular-targeting agents with different mechanisms of action can also be distinguished, and we find that drugs targeting only VEGFRs (Apatinib and Vandetanib) are not effective, whereas drugs that target VEGFRs, PDGFR and Tie2 (Linifanib and Cabozantinib) do regress the vasculature. Tumors in the VMT show strong metabolic heterogeneity when imaged using NADH Fluorescent Lifetime Imaging Microscopy and, compared to their surrounding stroma, many show a higher free/bound NADH ratio consistent with their known preference for aerobic glycolysis. The VMT platform provides a unique model for studying vascularized solid tumors in vitro.

Three-Dimensional Adult Cardiac Extracellular Matrix Promotes Maturation of Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes.

Pluripotent stem cell-derived cardiomyocytes (CMs) have great potential in the development of new therapies for cardiovascular disease. In particular, human induced pluripotent stem cells (iPSCs) may prove especially advantageous due to their pluripotency, their self-renewal potential, and their ability to create patient-specific cell lines. Unfortunately, pluripotent stem cell-derived CMs are immature, with characteristics more closely resembling fetal CMs than adult CMs, and this immaturity has limited their use in drug screening and cell-based therapies. Extracellular matrix (ECM) influences cellular behavior and maturation, as does the geometry of the environment-two-dimensional (2D) versus three-dimensional (3D). We therefore tested the hypothesis that native cardiac ECM and 3D cultures might enhance the maturation of iPSC-derived CMs in vitro. We demonstrate that maturation of iPSC-derived CMs was enhanced when cells were seeded into a 3D cardiac ECM scaffold, compared with 2D culture. 3D cardiac ECM promoted increased expression of calcium-handling genes, Junctin, CaV1.2, NCX1, HCN4, SERCA2a, Triadin, and CASQ2. Consistent with this, we find that iPSC-derived CMs in 3D adult cardiac ECM show increased calcium signaling (amplitude) and kinetics (maximum upstroke and downstroke) compared with cells in 2D. Cells in 3D culture were also more responsive to caffeine, likely reflecting an increased availability of calcium in the sarcoplasmic reticulum. Taken together, these studies provide novel strategies for maturing iPSC-derived CMs that may have applications in drug screening and transplantation therapies to treat heart disease.

Angiogenic sprouting is regulated by endothelial cell expression of Slug.

The Snail family of zinc-finger transcription factors are evolutionarily conserved proteins that control processes requiring cell movement. Specifically, they regulate epithelial-to-mesenchymal transitions (EMT) where an epithelial cell severs intercellular junctions, degrades basement membrane and becomes a migratory, mesenchymal-like cell. Interestingly, Slug expression has been observed in angiogenic endothelial cells (EC) in vivo, suggesting that angiogenic sprouting may share common attributes with EMT. Here, we demonstrate that sprouting EC in vitro express both Slug and Snail, and that siRNA-mediated knockdown of either inhibits sprouting and migration in multiple in vitro angiogenesis assays. We find that expression of MT1-MMP, but not of VE-Cadherin, is regulated by Slug and that loss of sprouting as a consequence of reduced Slug expression can be reversed by lentiviral-mediated re-expression of MT1-MMP. Activity of MMP2 and MMP9 are also affected by Slug expression, likely through MT1-MMP. Importantly, we find enhanced expression of Slug in EC in human colorectal cancer samples compared with normal colon tissue, suggesting a role for Slug in pathological angiogenesis. In summary, these data implicate Slug as an important regulator of sprouting angiogenesis, particularly in pathological settings.