Fundamentals and Translational Applications of Stem Cells and Biomaterials in Dental, Oral and Craniofacial Regenerative Medicine.

Regenerative dental medicine continuously expands to improve treatments for prevalent clinical problems in dental and oral medicine. Stem cell based translational opportunities include regenerative therapies for tooth restoration, root canal therapy, and inflammatory processes (e.g., periodontitis). The potential of regenerative approaches relies on the biological properties of dental stem cells. These and other multipotent somatic mesenchymal stem cell (MSC) types can in principle be applied as either autologous or allogeneic sources in dental procedures. Dental stem cells have distinct developmental origins and biological markers that determine their translational utility. Dental regenerative medicine is supported by mechanistic knowledge of the molecular pathways that regulate dental stem cell growth and differentiation. Cell fate determination and lineage progression of dental stem cells is regulated by multiple cell signaling pathways (e.g., WNTs, BMPs) and epigenetic mechanisms, including DNA modifications, histone modifications, and non-coding RNAs (e.g., miRNAs and lncRNAs). This review also considers a broad range of novel approaches in which stem cells are applied in combination with biopolymers, ceramics, and composite materials, as well as small molecules (agonistic or anti-agonistic ligands) and natural compounds. Materials that mimic the microenvironment of the stem cell niche are also presented. Promising concepts in bone and dental tissue engineering continue to drive innovation in dental and non-dental restorative procedures.

Scalable mesenchymal stem cell enrichment from bone marrow aspirate using deterministic lateral displacement (DLD) microfluidic sorting.

The growing interest in regenerative medicine has opened new avenues for novel cell therapies using stem cells. Bone marrow aspirate (BMA) is an important source of stromal mesenchymal stem cells (MSCs). Conventional MSC harvesting from BMA relies on archaic centrifugation methods, often leading to poor yield due to osmotic stress, high centrifugation force, convoluted workflow, and long experimental time (∼2-3 hours). To address these issues, we have developed a scalable microfluidic technology based on deterministic lateral displacement (DLD) for MSC isolation. This passive, label-free cell sorting method capitalizes on the morphological differences between MSCs and blood cells (platelets and RBCs) for effective separation using an inverted L-shaped pillar array. To improve throughput, we developed a novel multi-chip DLD system that can process 2.5 mL of raw BMA in 20 ± 5 minutes, achieving a 2-fold increase in MSC recovery compared to centrifugation methods. Taken together, we envision that the developed DLD platform will enable fast and efficient isolation of MSCs from BMA for effective downstream cell therapy in clinical settings.

Strategies to enhance immunomodulatory properties and reduce heterogeneity in mesenchymal stromal cells during ex vivo expansion.

Therapies using mesenchymal stromal cells (MSCs) to treat immune and inflammatory conditions are now at an exciting stage of development, with many MSC-based products progressing to phase II and III clinical trials. However, a major bottleneck in the clinical translation of allogeneic MSC therapies is the variable immunomodulatory properties of MSC products due to differences in their tissue source, donor heterogeneity and processes involved in manufacturing and banking. This variable functionality of MSC products likely contributes to the substantial inconsistency observed in the clinical outcomes of phase III trials of MSC therapies; several trials have failed to reach the primary efficacy endpoint. In this review, we discuss various strategies to consistently maintain or enhance the immunomodulatory potency of MSCs during ex vivo expansion, which will enable the manufacture of allogeneic MSC banks that have high potency and low variability. Biophysical and biochemical priming strategies, the use of culture additives such as heparan sulfates, and genetic modification can substantially enhance the immunomodulatory properties of MSCs during in vitro expansion. Furthermore, robust donor screening, the use of biomarkers to select for potent MSC subpopulations, and rigorous quality testing to improve the release criteria for MSC banks have the potential to reduce batch-to-batch heterogeneity and enhance the clinical efficacy of the final MSC product. Machine learning approaches to develop predictive models of individual patient response can enable personalized therapies and potentially establish correlations between in vitro potency measurements and clinical outcomes in human trials.

A combined cell and growth factor delivery for the repair of a critical size tibia defect using biodegradable hydrogel implants.

The ability to repair critical-sized long-bone injuries using growth factor and cell delivery was investigated using hydrogel biomaterials. Physiological doses of the recombinant human bone morphogenic protein-2 (rhBMP2) were delivered in a sustained manner from a biodegradable hydrogel containing peripheral human blood-derived endothelial progenitor cells (hEPCs). The biodegradable implants made from polyethylene glycol (PEG) and denatured fibrinogen (PEG-fibrinogen, PF) were loaded with 7.7 µg/ml of rhBMP2 and 2.5 × 106 cells/ml hEPCs. The safety and efficacy of the implant were tested in a rodent model of a critical-size long-bone defect. The hydrogel implants were formed ex-situ and placed into defects in the tibia of athymic nude rats and analyzed for bone repair after 13 weeks following surgery. The hydrogels containing a combination of 7.7 µg/ml of rhBMP2 and 2.5 × 106 cells/ml hEPCs were compared to control hydrogels containing 7.7 µg/ml of rhBMP2 only, 2.5 × 106 cells/ml hEPCs only, or bare hydrogels. Assessments of bone repair include histological analysis, bone formation at the site of implantation using quantitative microCT, and assessment of implant degradation. New bone formation was detected in all treated animals, with the highest amounts found in the treatments that included animals that combined the PF implant with rhBMP2. Moreover, statistically significant increases in the tissue mineral density (TMD), trabecular number and trabecular thickness were observed in defects treated with rhBMP2 compared to non-rhBMP2 defects. New bone formation was significantly higher in the hEPC-treated defects compared to bare hydrogel defects, but there were no significant differences in new bone formation, trabecular number, trabecular thickness or TMD at 13 weeks when comparing the rhBMP2 + hEPCs-treated defects to rhBMP2-treated defects. The study concludes that the bone regeneration using hydrogel implants containing hEPCs are overshadowed by enhanced osteogenesis associated with sustained delivery of rhBMP2.

FGFR2 accommodates osteogenic cell fate determination in human mesenchymal stem cells.

The multilineage differentiation potential of human mesenchymal stem cells (hMSCs) underpins their clinical utility for tissue regeneration. Control of such cell-fate decisions is tightly regulated by different growth factors/cytokines and their cognate receptors. Fibroblast growth factors (FGFs) are among such factors critical for osteogenesis. However, how FGF receptors (FGFRs) help to orchestrate osteogenic progression remains to be fully elucidated. Here, we studied the protein levels of FGFRs during osteogenesis in human adult bone marrow-derived MSCs and discovered a positive correlation between FGFR2 expression and alkaline phosphatase (ALP) activity, an early marker of osteogenesis. Through RNA interference studies, we confirmed the role of FGFR2 in promoting the osteogenic differentiation of hMSCs. Knockdown of FGFR2 resulted in downregulation of pro-osteogenic genes and upregulation of pro-adipogenic genes and adipogenic commitment. Moreover, under osteogenic induction, FGFR2 knockdown resulted in upregulation of Enhancer of Zeste Homolog 2 (EZH2), an epigenetic enzyme that regulates MSC lineage commitment and suppresses osteogenesis. Lastly, we show that serial-passaged hMSCs have reduced FGFR2 expression and impaired osteogenic potential. Our study suggests that FGFR2 is critical for mediating osteogenic fate by regulating the balance of osteo-adipogenic lineage commitment. Therefore, examining FGFR2 levels during serial-passaging of hMSCs may prove useful for monitoring their multipotency.

Bone marrow mesenchymal stem cells with low dose bone morphogenetic protein 2 enhances scaffold-based spinal fusion in a porcine model.

High doses bone morphogenetic protein 2 (BMP-2) have resulted in a series of complications in spinal fusion. We previously established a polyelectrolyte complex (PEC) carrier system that reduces the therapeutic dose of BMP-2 in both rodent and porcine spinal fusion models. This study aimed to evaluate the safety and efficacy of the combination of bone marrow mesenchymal stem cells (BMSCs) and low dose BMP-2 delivered by PEC for bone regeneration in a porcine model of anterior lumbar interbody spinal fusion (ALIF) application. Six Yorkshire pigs underwent a tri-segmental (L2/L3; L3/L4; L4/L5) ALIF in four groups, namely: (a) BMSCs + 25 µg BMP-2/PEC (n = 9), (b) 25 µg BMP-2/PEC (n = 3), (c) BMSCs (n = 3), and (d) 50 µg BMP-2/absorbable collagen sponge (n = 3). Fusion outcomes were evaluated by radiography, biomechanical testing, and histological analysis after 12 weeks. Mean radiographic scores at 12 weeks were 2.7, 2.0, 1.0, and 1.0 for Groups 1 to 4, respectively. µ-CT scanning, biomechanical evaluation, and histological analysis demonstrated solid fusion and successful bone regeneration in Group 1. In contrast, Group 2 showed inferior quality and slow rate of fusion, and Groups 3 and 4 failed to fuse any of the interbody spaces. There was no obvious evidence of seroma formation, implant rejection, or any other complications in all groups. The results suggest that the combination of BMSCs and low dose BMP-2/PEC could further lower down the effective dose of the BMP-2 and be used as a bone graft substitute in the large animal ALIF model.

Fibroblastic differentiation of mesenchymal stem/stromal cells (MSCs) is enhanced by hypoxia in 3D cultures treated with bone morphogenetic protein 6 (BMP6) and growth and differentiation factor 5 (GDF5).

INTRODUCTION: Culture conditions and differentiation cocktails may facilitate cell maturation and extracellular matrix (ECM) secretion and support the production of engineered fibroblastic tissues with applications in ligament regeneration. The objective of this study is to investigate the potential of two connective tissue-related ligands (i.e., BMP6 and GDF5) to mediate collagenous ECM synthesis and tissue maturation in vitro under normoxic and hypoxic conditions based on the hypothesis that BMP6 and GDF5 are components of normal paracrine signalling events that support connective tissue homeostasis. METHODS: Human adipose-derived MSCs were seeded on 3D-printed medical-grade polycaprolactone (PCL) scaffolds using a bioreactor and incubated in media containing GDF5 and/or BMP6 for 21 days in either normoxic (5% oxygen) or hypoxic (2% oxygen) conditions. Constructs were harvested on Day 3 and 21 for cell viability analysis by live/dead staining, structural analysis by scanning electron microscopy, mRNA levels by RTqPCR analysis, and in situ deposition of proteins by immunofluorescence microscopy. RESULTS: Pro-fibroblastic gene expression is enhanced by hypoxic culture conditions compared to normoxic conditions. Hypoxia renders cells more responsive to treatment with BMP6 as reflected by increased expression of ECM mRNA levels on Day 3 with sustained expression until Day 21. GDF5 was not particularly effective either in the absence or presence of BMP6. CONCLUSIONS: Fibroblastic differentiation of MSCs is selectively enhanced by BMP6 and not GDF5. Environmental factors (i.e., hypoxia) also influenced the responsiveness of cells to this morphogen.

Age-Related Changes in the Inflammatory Status of Human Mesenchymal Stem Cells: Implications for Cell Therapy.

Human mesenchymal stem/stromal cell (hMSC)-based cell therapies are promising for treating a variety of diseases. The unique immunomodulatory properties of hMSCs have extended their therapeutic potential beyond tissue regeneration. However, extensive pre-clinical culture expansion inevitably drives cells toward replicative "aging" and a consequent decline in quality. These "in vitro-aged" hMSCs resemble biologically aged cells, which have been reported to show senescence signatures, diminished immunosuppressive capacity, and weakened regenerative potential as well as pro-inflammatory features. In this review, we have surveyed the literature to explore the intimate relationship between the inflammatory status of hMSCs and their in vitro aging process. We posit that a shift from an anti-inflammatory to a pro-inflammatory phenotype of culture-expanded hMSCs contributes to a deterioration in their therapeutic efficacy. Potential molecular and cellular mechanisms underpinning this phenomenon have been discussed. We have also highlighted studies that leverage these mechanisms to make culture-expanded hMSCs more amenable for clinical use.

Combination of BMP2 and EZH2 Inhibition to Stimulate Osteogenesis in a 3D Bone Reconstruction Model.

High concentrations of bone morphogenetic protein 2 (BMP2) in bone regeneration cause adverse events (e.g, heterotopic bone formation and acute inflammation). This study examines novel epigenetic strategies (i.e., EZH2 inhibition) for augmenting osteogenesis, thereby aiming to reduce the required BMP2 dose in vivo for bone regeneration and minimize these adverse effects. Human bone marrow-derived mesenchymal stem cells (BMSCs) were grown on three-dimensional (3D)-printed medical-grade polycaprolactone scaffolds and incubated in osteogenic media containing 50 ng/mL BMP2 and/or 5 µM GSK126 (EZH2 inhibitor) for 6 days (n = 3 per group and timepoint). Constructs were harvested for realtime quantitative polymerase chain reaction analysis at Day 10 and immunofluorescence (IF) microscopy at Day 21. After pretreating for 6 days and maintaining in osteogenic media for 4 days, BMSC-seeded scaffolds were also implanted in an immunocompromised subcutaneous murine model (n = 39; 3/group/donor and 3 control scaffolds) for histological analysis at 8 weeks. Pretreatment of BMSCs with BMP2 and BMP2/GSK126 costimulated expression of osteoblast-related genes (e.g., IBSP, SP7, RUNX2, and DLX5), as well as protein accumulation (e.g., collagen type 1/COL1A1 and osteocalcin/BGLAP) based on IF staining. While in vivo implantation for 8 weeks did not result in bone formation, increased angiogenesis was observed in BMP2 and BMP2/GSK126 groups. This study finds that BMP2 and GSK126 costimulate osteogenic differentiation of MSCs on 3D scaffolds in vitro and may contribute to enhanced vascularization when implanted in vivo to support bone formation. Thus, epigenetic priming with EZH2 inhibitors may have translational potential in bone healing by permitting a reduction of BMP2 dosing in vivo to mitigate its side effects. Impact statement While autografts are still the gold standard for bone reconstruction, tissue availability and donor morbidity are significant limitations. Previous attempts to use high concentrations of bone morphogenetic protein 2 (BMP2) have been shown to cause adverse events such as excessive bone formation and acute inflammation. Overall, the utilization of EZH2 inhibitors to modulate gene expression in favor of bone healing has been demonstrated in vitro in a tissue engineering strategy. Our study will pave the way to developing tissue engineering strategies involving GSK126 as an adjuvant to increase the effects of BMP2 for stimulating cells of interest on a three-dimensional scaffold for bone regeneration.

Heparan Sulfate Proteoglycans: Key Mediators of Stem Cell Function.

Heparan sulfate proteoglycans (HSPGs) are an evolutionarily ancient subclass of glycoproteins with exquisite structural complexity. They are ubiquitously expressed across tissues and have been found to exert a multitude of effects on cell behavior and the surrounding microenvironment. Evidence has shown that heterogeneity in HSPG composition is crucial to its functions as an essential scaffolding component in the extracellular matrix as well as a vital cell surface signaling co-receptor. Here, we provide an overview of the significance of HSPGs as essential regulators of stem cell function. We discuss the various roles of HSPGs in distinct stem cell types during key physiological events, from development through to tissue homeostasis and regeneration. The contribution of aberrant HSPG production to altered stem cell properties and dysregulated cellular homeostasis characteristic of cancer is also reviewed. Finally, we consider approaches to better understand and exploit the multifaceted functions of HSPGs in influencing stem cell characteristics for cell therapy and associated culture expansion strategies.

Vascular Endothelial Growth Factor 165-Binding Heparan Sulfate Promotes Functional Recovery From Cerebral Ischemia.

BACKGROUND AND PURPOSE: Although VEGF165 (vascular endothelial growth factor-165) is able to enhance both angiogenesis and neurogenesis, it also increases vascular permeability through the blood-brain barrier. Heparan sulfate (HS) sugars play important roles in regulating VEGF bioactivity in the pericellular compartment. Here we asked whether an affinity-purified VEGF165-binding HS (HS7) could augment endogenous VEGF activity during stroke recovery without affecting blood-brain barrier function. METHODS: Both rat brain endothelial cell line 4 and primary rat neural progenitor cells were used to evaluate the potential angiogenic and neurogenic effects of HS7 in vitro. For in vivo experiments, male Sprague-Dawley rats were subjected to 100 minutes of transient focal cerebral ischemia, then treated after 4 days with either PBS or HS7. One week later, infarct volume, behavioral sequelae, immunohistochemical markers of angiogenesis and neural stem cell proliferation were assessed. RESULTS: HS7 significantly enhanced VEGF165-mediated angiogenesis in rat brain endothelial cell line 4 brain endothelial cells, and increased the proliferation and differentiation of primary neural progenitor cells, both via the VEGFR2 (vascular endothelial growth factor receptor 2) pathway. Intracerebroventricular injection of HS7 improved neurological outcome in ischemic rats without changing infarct volumes. Immunostaining of the compromised cerebrum demonstrated increases in collagen IV/Ki67 and nestin/Ki67 after HS7 exposure, consistent with its ability to promote angiogenesis and neurogenesis, without compromising blood-brain barrier integrity. CONCLUSIONS: A VEGF-activating glycosaminoglycan sugar, by itself, is able to enhance endogenous VEGF165 activity during the post-ischemic recovery phase of stroke.

A genomic biomarker that identifies human bone marrow-derived mesenchymal stem cells with high scalability.

Although the application of human mesenchymal stem cells (hMSCs) to repair damaged or diseased tissues has proven relatively effective, both the donor-to-donor variability in ex vivo expansion rates and the maintenance of stemness remain a bottleneck to widespread translation. Previous work from this laboratory stratified donors into those yielding hMSCs with high- or low-growth capacity; global transcriptomic analysis revealed that high-growth-capacity hMSCs were characterized by a loss of the gene encoding glutathione S-transferase theta 1 (GSTT1). These GSTT1-null hMSCs demonstrated increased proliferative rates, clonogenic potential, and longer telomeres compared with low-growth capacity hMSCs that were GSTT1-positive. Thus, this study identifies GSTT1 as a novel genomic DNA biomarker for hMSC scalability.

Ascorbate and Iron Are Required for the Specification and Long-Term Self-Renewal of Human Skeletal Mesenchymal Stromal Cells.

The effects of ascorbate on adult cell fate specification remain largely unknown. Using our stepwise and chemically defined system to derive lateral mesoderm progenitors from human pluripotent stem cells (hPSCs), we found that ascorbate increased the expression of mesenchymal stromal cell (MSC) markers, purity of MSCs, the long-term self-renewal and osteochondrogenic capacity of hPSC-MSCs in vitro. Moreover, ascorbate promoted MSC specification in an iron-dependent fashion, but not in a redox-dependent manner. Further studies revealed that iron synergized with ascorbate to regulate hPSC-MSC histone methylation, promote their long-term self-renewal, and increase their osteochondrogenic capacity. We found that one of the histone demethylases affected by ascorbate, KDM4B, was necessary to promote the specification of hPSC-MSCs. This mechanistic understanding led to the metabolic optimization of hPSC-MSCs with an extended lifespan in vitro and the ability to fully repair cartilage defects upon transplantation in vivo. Our results highlight the importance of ascorbate and iron metabolism in adult human cell fate specification.

Enhancing the Efficacy of Stem Cell Therapy with Glycosaminoglycans.

Human 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.

Bringing Heparan Sulfate Glycomics Together with Proteomics for the Design of Novel Therapeutics: A Historical Perspective.

Increasing knowledge of how peptides bind saccharides, and of how saccharides bind peptides, is starting to revolutionize understanding of cell-extracellular matrix relationships. Here, a historical perspective is taken of the relationship between heparan sulfate glycosaminoglycans and how they interact with peptide growth factors in order to both drive and modulate signaling through the appropriate cognate receptors. Such knowledge is guiding the preparation of targeted sugar mimetics that will impact the treatment of many different kinds of diseases, including cancer.

A polycaprolactone-ß-tricalcium phosphate-heparan sulphate device for cranioplasty.

BACKGROUND: Cranioplasty is a surgical procedure used to treat a bone defect or deformity in the skull. To date, there is little consensus on the standard-of-care for graft materials used in such a procedure. Graft materials must have sufficient mechanical strength to protect the underlying brain as well as the ability to integrate and support new bone growth. Also, the ideal graft material should be individually customized to the contours of the defect to ensure a suitable aesthetic outcome for the patient. PURPOSE: Customized 3D-printed scaffolds comprising of polycaprolactone-ß-tricalcium phosphate (PCL-TCP) have been developed with mechanical properties suitable for cranioplasty. Osteostimulation of PCL-TCP was enhanced through the addition of a bone matrix-mimicking heparan sulphate glycosaminoglycan (HS3) with increased affinity for bone morphogenetic protein-2 (BMP-2). Efficacy of this PCL-TCP/HS3 combination device was assessed in a rat critical-sized calvarial defect model. METHOD: Critical-sized defects (5 mm) were created in both parietal bones of 19 Sprague Dawley rats (Male, 450-550 g). Each cranial defect was randomly assigned to 1 of 4 treatment groups: (1) A control group consisting of PCL-TCP/Fibrin alone (n = 5); (2) PCL-TCP/Fibrin-HSft (30 µg) (n = 6) (HSft is the flow-through during HS3 isolation that has reduced affinity for BMP-2); (3) PCL-TCP/Fibrin-HS3 (5 µg) (n = 6); (4) PCL-TCP/Fibrin-HS3 (30 µg) (n = 6). Scaffold integration and bone formation was evaluated 12-weeks post implantation by µCT and histology. RESULTS: Treatment with PCL-TCP/Fibrin alone (control) resulted in 23.7% ± 1.55% (BV/TV) of the calvarial defect being filled with new bone, a result similar to treatment with PCL-TCP/Fibrin scaffolds containing either HSft or HS3 (5 µg). At increased amounts of HS3 (30 µg), enhanced bone formation was evident (BV/TV = 38.6% ± 9.38%), a result 1.6-fold higher than control. Further assessment by 2D µCT and histology confirmed the presence of enhanced bone formation and scaffold integration with surrounding host bone only when scaffolds contained sufficient bone matrix-mimicking HS3. CONCLUSION: Enhancing the biomimicry of devices using a heparan sulphate with increased affinity to BMP-2 can serve to improve the performance of PCL-TCP scaffolds and provides a suitable treatment for cranioplasty.

Validation of Osteogenic Properties of Cytochalasin D by High-Resolution RNA-Sequencing in Mesenchymal Stem Cells Derived from Bone Marrow and Adipose Tissues.

Differentiation of mesenchymal stromal/stem cells (MSCs) involves a series of molecular signals and gene transcription events required for attaining cell lineage commitment. Modulation of the actin cytoskeleton using cytochalasin D (CytoD) drives osteogenesis at early timepoints in bone marrow-derived MSCs and also initiates a robust osteogenic differentiation program in adipose tissue-derived MSCs. To understand the molecular basis for these pronounced effects on osteogenic differentiation, we investigated global changes in gene expression in CytoD-treated murine and human MSCs by high-resolution RNA-sequencing (RNA-seq) analysis. A three-way bioinformatic comparison between human adipose tissue-derived MSCs (hAMSCs), human bone marrow-derived MSCs (hBMSCs), and mouse bone marrow-derived MSCs (mBMSCs) revealed significant upregulation of genes linked to extracellular matrix organization, cell adhesion and bone metabolism. As anticipated, the activation of these differentiation-related genes is accompanied by a downregulation of nuclear and cell cycle-related genes presumably reflecting cytostatic effects of CytoD. We also identified eight novel CytoD activated genes-VGLL4, ARHGAP24, KLHL24, RCBTB2, BDH2, SCARF2, ACAD10, HEPH-which are commonly upregulated across the two species and tissue sources of our MSC samples. We selected the Hippo pathway-related VGLL4 gene, which encodes the transcriptional co-factor Vestigial-like 4, for further study because this pathway is linked to osteogenesis. VGLL4 small interfering RNA depletion reduces mineralization of hAMSCs during CytoD-induced osteogenic differentiation. Together, our RNA-seq analyses suggest that while the stimulatory effects of CytoD on osteogenesis are pleiotropic and depend on the biological state of the cell type, a small group of genes including VGLL4 may contribute to MSC commitment toward the bone lineage.

Immobilization of vitronectin-binding heparan sulfates onto surfaces to support human pluripotent stem cells.

Functionalizing medical devices with polypeptides to enhance their performance has become important for improved clinical success. The extracellular matrix (ECM) adhesion protein vitronectin (VN) is an effective coating, although the chemistry used to attach VN often reduces its bioactivity. In vivo, VN binds the ECM in a sequence-dependent manner with heparan sulfate (HS) glycosaminoglycans. We reasoned therefore that sequence-based affinity chromatography could be used to isolate a VN-binding HS fraction (HS9) for use as a coating material to capture VN onto implant surfaces. Binding avidity and specificity of HS9 were confirmed by enzyme-linked immunosorbent assay (ELISA) and surface plasmon resonance (SPR)-based assays. Plasma polymerization of allylamine (AA) to tissue culture-treated polystyrene (TCPS) was then used to capture and present HS9 as determined by radiolabeling and ELISA. HS9-coated TCPS avidly bound VN, and this layered surface supported the robust attachment, expansion, and maintenance of human pluripotent stem cells. Compositional analysis demonstrated that 6-O- and N-sulfation, as well as lengths greater than three disaccharide units (dp6) are critical for VN binding to HS-coated surfaces. Importantly, HS9 coating reduced the threshold concentration of VN required to create an optimally bioactive surface for pluripotent stem cells. We conclude that affinity-purified heparan sugars are able to coat materials to efficiently bind adhesive factors for biomedical applications. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 106B: 1887-1896, 2018.

Osteogenic Stimulation of Human Adipose-Derived Mesenchymal Stem Cells Using a Fungal Metabolite That Suppresses the Polycomb Group Protein EZH2.

Strategies for musculoskeletal tissue regeneration apply adult mesenchymal stem/stromal cells (MSCs) that can be sourced from bone marrow- and lipo-aspirates. Adipose tissue-derived MSCs are more easily harvested in the large quantities required for skeletal tissue-engineering approaches, but are generally considered to be less osteogenic than bone marrow MSCs. Therefore, we tested a new molecular strategy to improve their osteogenic lineage-differentiation potential using the fungal metabolite cytochalasin D (CytoD). We show that CytoD, which may function by redistributing the intracellular location of ß-actin (ACTB), is a potent osteogenic stimulant as reflected by significant increases in alkaline phosphatase activity, extracellular matrix mineralization, and osteoblast-related gene expression (e.g., RUNX2, ALPL, SPARC, and TGFB3). RNA sequencing analyses of MSCs revealed that acute CytoD treatment (24 hours) stimulates a broad program of osteogenic biomarkers and epigenetic regulators. CytoD decreases mRNA and protein levels of the Polycomb chromatin regulator Enhancer of Zeste Homolog 2 (EZH2), which controls heterochromatin formation by mediating trimethylation of histone 3 lysine 27 (H3K27me3). Reduced EZH2 expression decreases cellular H3K27me3 marks indicating a global reduction in heterochromatin. We conclude that CytoD is an effective osteogenic stimulant that mechanistically functions by blocking both cytoplasmic actin polymerization and gene-suppressive epigenetic mechanisms required for the acquisition of the osteogenic phenotype in adipose tissue-derived MSCs. This finding supports the use of CytoD in advancing the osteogenic potential of MSCs in skeletal regenerative strategies. Stem Cells Translational Medicine 2018;7:197-209.