Presence Of Matrigel Research Articles

Human Metastatic Melanoma Cell Lines Panel for In Vitro and In Vivo Investigations

The melanoma origin of cell lines obtained from the axillary lymph node (mel Kas, mel Pet, and mel Lap from patients with a verified diagnosis) was confirmed by the detection of the Melan A melanocyte marker expression. A hyperdiploid (2n+) for the mel Kas line; near-diploid (2n), and in some cells near-tertaploid (4n), and even hypo-octaploid (8n) set (172–179 chromosomes) in the mel Pet cell line; and a hypotetraploid (4n−) for the mel Lap line were detected by karyotypic analysis. All three cell lines are tumorigenic; however, mel Pet demonstrates tumor growth in Balb/c nude mice only in the presence of matrigel. All three lines showed a high expression of TUBB3 and PD-L1 markers, while ERa was low (minimum for mel Pet). Significant differences in the expression level were shown for the Cyt molecular marker. In the transplantation of cells to Balb/c nude mice, a stable expression level is observed only for TUBB3. For the rest of the markers, a decrease in their expression level of varying degrees was noted when the cells were growing in solid tumors in vivo. Mutations were detected in oncogenes (BRAF, EZH2, KIT, KRAS, NRAS, ROS1) and tumor suppressor genes (CDKN2A, FAT4, KMT2C, LRP1B, PTEN, PTPRB, TP53). The detailed characterization of the cell lines makes them valuable for various scientific and regulatory experiments, particularly those involving preclinical data on antiproliferative drugs for malignant melanoma or investigations into melanoma cell properties and progression.

Cancer-associated fibroblasts as cellular vehicles in endometrial cancer cell migration.

Cell motility is a critical step in the metastasis cascade. However, the role of cancer-associated fibroblasts (CAFs) in facilitating endometrial cancer (EC) cell motility remains unclear. The present study aimed to investigate the role of CAFs in EC motility in a 3D environment. A co-culture model was established using an EC cell line (ECC-1) and CAFs on a Matrigel® matrix and compared to the respective individual monocultures. It was demonstrated that endometrial CAFs increased the motility of the EC cell line, compared with the monoculture. Using live cell imaging, CAFs were observed to form cell projections that served as contact guidance for ECC-1 cell locomotion in the spheroid formation process. These effects were specific to CAFs, as fibroblasts isolated from benign endometrial tissue samples did not form cell projections. Molecular analysis revealed that RhoA/Rho-associated, coiled-coil containing protein kinase 1 (ROCK1) signaling activation partly contributed to CAF-mediated ECC-1 cell migration. The presence of Matrigel® increased the mRNA expression of RhoA, and the mRNA and protein expression levels of its downstream effectors, ROCK1 and p-MLC, respectively, in the ECC-1 and CAF co-culture, as well as the ECC-1 and CAF monocultures. Interestingly, high phosphorylation levels of myosin light chain mediated the activation of RhoA/ROCK1 signaling in the ECC-1 and CAF co-culture. The ROCK1 inhibitor Y-27632 attenuated the motility of tumor cells in ECC-1 and CAF co-cultures. However, similar treatment led to a significant inhibition in the motility of the CAF monoculture, but not the ECC-1 monoculture. Moreover, tumor spheroid formation was inhibited due to a reduction in stress fiber formation in ECC-1 and CAF co-cultures. Altogether, these findings suggest that the regulation of the RhoA/ROCK1 signaling pathway is required for CAFs to serve as cellular vehicles in order for EC cells to migrate and form spheroids in a 3D environment.

Abstract 3150: A characterization of cancer vasculogenic mimicry: Extracellular matrix induced cellular signaling to lumen formation

Abstract Introduction: Vasculogenic mimicry (VM) describes a process by which cancer cells establish an alternative perfusion pathway in an endothelial cell-free manner. Despite the strong correlation with reduced patient survival, the mechanisms by which a tumor can create this self-generated irrigation system are still not fully understood. The process of VM in vitro can only occur in Matrigel; however, the protein component and signaling pathways involved in this process are unknown. Methods: Using an established in vitro model of VM, of ovarian and breast cancer cells (HEY and MDA-MB-231, respectively) on Matrigel coating, we utilized pharmacological inhibitors, gene silencing and blocking antibodies to elucidate the signaling pathways involved in the process of VM. Immunofluorescence and sirius red staining were used to determine the glycoprotein-rich component lining the lumen of the tubular structures. Results: Differently to what observed in the presence of Matrigel, VM did not occur when cancer cells were cultivated on plastic, glass or heat denatured Matrigel (10 mins at 65°C). Using exclusively Collagen I or Laminin 111 to mimic the extracellular matrix we observed than only in the presence of Laminin 111 could VM formation occur. Laminin is secreted and deposited by HEY cells and constitutes a part of the luminal lining. Silencing of integrin β1, but not β3, by siRNA and antibody blocking prevents this process. Chemical inhibition of PI3K pathway and metalloproteases (MMP) activation demonstrate that these pathways are also essential. RNAseq analysis suggests that this process has minimal dependence on de novo transcriptional activity. Discussion and conclusion: We have shown that VM only occurs when cells are seeded on Matrigel but not on plastic, glass or heat denatured Matrigel, suggesting that this phenomenon is susceptible to substrate/matrix rigidity. Furthermore, we identified Laminin as the essential matrix protein secreted and deposited by cancer cells to allow for VM assembly. Its interaction with integrin β1, and the consequent regulation of the activity of MMP leads to the remodeling of the ECM to favor the connection of the VM channels to the microcirculation system. This pathway is not heavily dependent on transcription but requires the PI3K pathway. As VM is strongly associated with poor patient survival, understanding the formation of this alternative irrigation system may deliver new druggable targets. Citation Format: Gabriel Mingo, Andres Valdivia, Varina Aldana, Javiera Pradenas, Nicole Babbitt, Pamela Gonzalez, Francisco Nualart, Jorge Díaz, Lisette Leyton, Cristina Bertocchi, Gareth Owen. A characterization of cancer vasculogenic mimicry: Extracellular matrix induced cellular signaling to lumen formation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 3150.

The role of nonlinear mechanical properties of biomimetic hydrogels for organoid growth.

Cell-driven plastic remodeling of the extracellular matrix (ECM) is a key regulator driving cell invasion and organoid morphogenesis in 3D. While, mostly, the linear properties are reported, the nonlinear and plastic property of the used matrix is required for these processes to occur. Here, we report on the nonlinear and plastic mechanical properties of networks derived from collagen I, Matrigel, and related hybrid gels and link their mechanical response to the underlying collagen structure. We reveal the predominantly linear behavior of Matrigel over a wide range of strains and contrast this to the highly nonlinear and plastic response of collagen upon mechanical load. We show that the mechanical nonlinear response of collagen can be gradually diminished by enriching the network stepwise with Matrigel. This tunability results from the suppression of collagen polymerization in the presence of Matrigel, resulting in a collagen network structure with significant smaller mesh size and consequent contribution to the mechanical response. Thus, the nonlinear plastic properties and structure of the ECM is not simply the addition of two independent network types but depends on the exact polymerization conditions. The understanding of this interplay is key toward an understanding of the dependencies of cellular interactions with their ECM and sheds light on the nonlinear cell-ECM interaction during organogenesis.

Per- and Polyfluoroalkyl Substances Differentially Inhibit Placental Trophoblast Migration and Invasion InVitro.

Per- and polyfluoroalkyl substances (PFAS) are used as industrial surfactants and chemical coatings for household goods such as Teflon. Despite regulatory efforts to phase out legacy PFAS, they remain detectable in drinking water throughout the United States. This is due to the stability of legacy PFAS and the continued use of replacement compounds. In humans, PFAS have been detected in placenta and cord blood and are associated with low birth weight and preeclampsia risk. Preeclampsia is a leading cause of maternal mortality and is driven by insufficient endometrial trophoblast invasion, resulting in poor placental blood flow. PFAS alter invasion of other cell types, but their impact on trophoblasts is not understood. We therefore assessed the effects of PFAS on trophoblast migration, invasion, and gene expression in vitro. Trophoblast migration and invasion were assessed using a modified scratch assay in the absence or presence of Matrigel, respectively. Treatment with perfluorooctanoic sulfate (PFOS), perfluorooctanoic acid (PFOA), and GenX (1000 ng/ml) each decreased trophoblast migration over 24 h. However, only GenX (1000 ng/ml) significantly inhibited trophoblast invasion. Treatment with PFOS, PFOA, and GenX also decreased trophoblast expression of chemokines (eg, CCL2), chemokine receptors (eg, CCR4), and inflammatory enzymes (eg, ALOX15) involved in migration. Inhibition of chemokine receptors with pertussis toxin (10 ng/ml), a G-protein inhibitor, inhibited trophoblast migration similar to the PFAS. Taken together, PFAS decrease trophoblast migration, invasion, and inflammatory signaling. By understanding the mechanisms involved, it may be possible to identify the biological and exposure factors that contribute to preeclampsia.

Human fallopian tube epithelial cells exhibit stemness features, self-renewal capacity, and Wnt-related organoid formation

BackgroundFallopian tube epithelial cells (FTEC) were thought to be the origin of high-grade serous ovarian carcinoma (HGSOC). Knowledge of the stemness or initiating characteristics of FTEC is insufficient. Previously, we have characterized the stemness cell marker of FTEC, this study aims to further characterize the clonogenicity and spheroid features of FTEC.MethodsWe successfully derived FTECs from the epithelial layer of the human fallopian tubes. We examined the morphology, proliferation rate, doubling time, and clonal growth of them. At passage 3, the sphere formations on gelatin-coated culture, suspension culture, and matrigel culture were observed, and the expression of LGR5, SSEA3, SSEA4, and other stemness markers was examined. Furthermore, tissue-reconstituted organoids from coculture of FTEC, fallopian stromal cells (FTMSC) and endothelial cells (HUVEC) were examined.ResultsFTEC exhibited cuboidal cell morphology and maintained at a constant proliferation rate for up to nine passages (P9). FTEC could proliferate from a single cell with a clonogenic efficiency of 4%. Flow cytometry revealed expressions of normal stem cell markers (SSEA3, SSEA4, and LGR5) and cancer stem cell markers (CD24, CD44, CD117, ROR1, and CD133). FTEC formed spheres and colonies when cultured on low attach dish. In the presence of Matrigel, the stemness and colony formation activity were much enhanced. In co-culturing with FTMSC and HUVEC, FTEC could form organoids that could be blocked by Wnt inhibitor DKK1. Expressions of LGR5 and FOXJ1 expression were also decreased by adding DKK1.ConclusionWe demonstrated abundantly presence of stem cells in human FTECs which are efficient in forming colonies, spheres and organoids, relying on Wnt signaling. We also reported for the first time the generation of organoid from reconstitutied cell lineages in the tissue. This may provide a new model for studying the regneration and malignant transformation of the tubal epithelium.

Influence of Matrigel on Single- and Multiple-Spheroid Cultures in Breast Cancer Research

This study aimed to develop and compare single and multiple 3D models such as multicellular tumor spheroids and to investigate the influence of Matrigel on their morphological and functional behavior. MDA-MB-231 3D models were generated in the presence and absence of Matrigel and their key biological properties within 6 days of culture were monitored. Our results revealed the formation of well-defined 3D models in the presence of Matrigel, with a uniform morphology, increased diameter, good circularity, and increased expression of a proliferation marker (PCNA). In comparison, 3D models generated without Matrigel were characterized by an irregular border, reduced dimensions and circularity, and a decrease of PCNA expression. Similarities between the single and multiple 3D cultures were found in their viability, Nrf2 expression, and glutathione (GSH) content. The influence of Matrigel on MDA-MB-231 spheroids metabolism under hypoxic conditions was highlighted by released lactate dehydrogenase and nitric oxide, GSH levels and expression of Nrf2 and Hsp70 proteins. Based on the increased expression of PCNA and the development of the hypoxia process in the presence of extracellular matrix, our study showed that the addition of Matrigel improves the growing environment of tumor spheroids, making it closer to that of in vivo tumor conditions.

Complete Disaggregation of MCF-7-derived Breast Tumour Spheroids with Very Low Concentrations of α-Mangostin Loaded in CD44 Thioaptamer-tagged Nanoparticles.

Background: α-Mangostin (αMG) is a natural substance that exerts a wide range of antitumor effects. Recently, we described that free αMG was able to dissociate multicellular tumour spheroids (MCTSs) generated from breast carcinoma cells and to reduce their cellular viability and motility. Here, αMG was encapsulated into lipidic nanoparticles (NPs), conjugated or not to a CD44 thioaptamer, and the anticancer action evaluated against MCF-7 breast MCTSs.Methods: NPs containing αMG were formulated with a core of polylactic-co-glycolyc acid. Some of them were decorated with a CD44 thioaptamer using as catalysts 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide and N-hydroxysuccinimide. Both size and density of MCF-7-derived MCTSs were monitored during 72 h of treatment with NPs carrying 0.1, 0.5 and 1.0 μg/ml final concentrations of αMG. MCTSs were cultured on Matrigel or gelatine to better simulate the extracellular environment.Results: The NPs without thioaptamer and conveying 0.1 μg/ml αMG caused a significant dissociation of the MCTSs grown in gelatine after 24 h of treatment (p < 0.01). The most significant disaggregation of MCTSs was obtained using NPs carrying 0.5 μg/ml αMG (p < 0.01). A similar dissociating effect was observed when MCTSs were cultured in Matrigel under the same conditions for 48 - 72 h. By contrast, only concentrations over 1.0 μg/ml of free αMG were able to provoke a damage to MCTSs, consisting in a substantial reduction in their size (p < 0.05). Since the MCTS dissociation induced by αMG-loaded NPs occurred only in the presence of Matrigel or gelatine, an impairment of cell contacts to collagen fibres was likely responsible of this effect. Finally, the treatment of MCTSs with αMG-loaded NPs that were conjugated to the CD44 thioaptamer caused a similar decrease in density but a lower expansion of the spheroid, suggesting that a significant number of cells were died or arrested in cycle.Conclusion: Very low concentrations of αMG delivered by lipidic NPs are sufficient to provoke a substantial disaggregation of MCF-7 MCTSs that involves cell-to-collagen contacts. Similarly, the treatment of MCTSs with NPs conjugated to a CD44 thioaptamer leads to MCTS dissociation but through a more damaging action that causes also a reduction in cell number.

Generation of Otic Sensory Neurons from Mouse Embryonic Stem Cells in 3D Culture.

The peripheral hearing process taking place in the cochlea mainly depends on two distinct sensory cell types: the mechanosensitive hair cells and the spiral ganglion neurons (SGNs). The first respond to the mechanical stimulation exerted by sound pressure waves on their hair bundles by releasing neurotransmitters and thereby activating the latter. Loss of these sensorineural cells is associated with permanent hearing loss. Stem cell-based approaches aiming at cell replacement or in vitro drug testing to identify potential ototoxic, otoprotective, or regenerative compounds have lately gained attention as putative therapeutic strategies for hearing loss. Nevertheless, they rely on efficient and reliable protocols for the in vitro generation of cochlear sensory cells for their implementation. To this end, we have developed a differentiation protocol based on organoid culture systems, which mimics the most important steps of in vivo otic development, robustly guiding mouse embryonic stem cells (mESCs) toward otic sensory neurons (OSNs). The stepwise differentiation of mESCs toward ectoderm was initiated using a quick aggregation method in presence of Matrigel in serum-free conditions. Non-neural ectoderm was induced via activation of bone morphogenetic protein (BMP) signaling and concomitant inhibition of transforming growth factor beta (TGFβ) signaling to prevent mesendoderm induction. Preplacodal and otic placode ectoderm was further induced by inhibition of BMP signaling and addition of fibroblast growth factor 2 (FGF2). Delamination and differentiation of SGNs was initiated by plating of the organoids on a 2D Matrigel-coated substrate. Supplementation with brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3) was used for further maturation until 15 days of in vitro differentiation. A large population of neurons with a clear bipolar morphology and functional excitability was derived from these cultures. Immunostaining and gene expression analysis performed at different time points confirmed the transition trough the otic lineage and final expression of the key OSN markers. Moreover, the stem cell-derived OSNs exhibited functional electrophysiological properties of native SGNs. Our established in vitro model of OSNs development can be used for basic developmental studies, for drug screening or for the exploration of their regenerative potential.

Abstract 1490: Matrigel rescues breast cancer cells from the growth inhibitory effects of HIF-1α and HIF-2α silencing

Abstract Tumor microenvironments are frequently hypoxic and result in the stabilization of hypoxia inducible factors (HIF-1/2) that transcriptionally activate genes involved in invasion, metastasis, metabolism and angiogenesis [1]. The role of hypoxia and the contribution of HIF in the angiogenic switch leading to tumor progression and resistance to treatment are well documented. This angiogenic response to HIF activity is largely mediated through activation of vascular endothelial growth factor (VEGF). Noninvasive characterization of the loss of both isoform of HIFs (HIF-1α &amp; HIF-2α) on tumor vascularization is relatively unexplored. Here we investigated the effect of HIF silencing on tumor growth in the presence or absence of Matrigel that resembles the complex extracellular matrix (ECM) found in most tumors and determined its effect on tumor vasculature using noninvasive MRI. MDA-MB-231 human breast cancer cells expressing shRNA against both HIF-1α and HIF-2α (231-DK) were established as previously described [2]. In vivo studies were performed using MDA-MB-231 breast cancer cells expressing an empty vector control (231-EV) and 231-DK cells implanted in the mammary fat pad of female SCID mice. Tumor growth curves were obtained from cells inoculated either in 0.05 ml of Hanks balanced salt solution (HBSS) or together with Matrigel solution (8.8 mg/ml). All MRI studies were performed on a 9.4T Bruker Biospec horizontal bore scanner. 3D maps of vascular volume (V/V) and permeability (VP) were obtained using a rapid gradient-echo sequence and albumin-GdDTPA (0.5 g/kg) as the contrast agent (CA). A proton density (PD) image was acquired prior to CA injection, using a 3D gradient echo sequence, with TE/TR = 1.5/10 ms and 3° flip angle and analysis of the images were performed as previously described [3]. Exposure to hypoxia showed no increase in HIF-1 or 2α protein expression in 231-DK compared to 231-EV cells. A significant growth advantage of the 231-DK cells in vivo was observed when inoculated in the presence of Matrigel compared to 231-DK in HBSS. Growth advantage of tumors in the presence of Matrigel was less dramatic for 231-EV cells. When inoculated with Matrigel, 3D reconstructed maps of 231-DK tumors showed significantly increased VP compared to 231-EV tumors with no difference in the VV. Increased vascular permeability in tumors derived from 231-DK cells compared to tumors derived from 231-EV cells can be attributed to VEGF in the Matrigel that is known to exhibit paracrine effects. These data suggest that ECM components may modulate molecular targeting and highlight the importance of the tumor microenvironment in modifying HIF silencing effects. Work is under way to analyze the effects on the metastatic burden in these systems.

Extracellular Matrix can Recover the Downregulation of Adhesion Molecules after Cell Detachment and Enhance Endothelial Cell Engraftment.

The low cell engraftment after transplantation limits the successful application of stem cell therapy and the exact pathway leading to acute donor cell death following transplantation is still unknown. Here we investigated if processes involved in cell preparation could initiate downregulation of adhesion-related survival signals, and further affect cell engraftment after transplantation. Human embryonic stem cell-derived endothelial cells (hESC-ECs) were suspended in PBS or Matrigel and kept at 4 °C. Quantitative RT-PCR analysis was used to test the adhesion and apoptosis genes’ expression of hESC-ECs. We demonstrated that cell detachment can cause downregulation of cell adhesion and extracellular matrix (ECM) molecules, but no obvious cell anoikis, a form of apoptosis after cell detachment, was observed. The downregulation of adhesion and ECM molecules could be regained in the presence of Matrigel. Finally, we transplanted hESC-ECs into a mouse myocardial ischemia model. When transplanted with Matrigel, the long-term engraftment of hESC-ECs was increased through promoting angiogenesis and inhibiting apoptosis, and this was confirmed by bioluminescence imaging. In conclusion, ECM could rescue the functional genes expression after cell detached from culture dish, and this finding highlights the importance of increasing stem cell engraftment by mimicking stem cell niches through ECM application.

Controlled 3D culture in Matrigel microbeads to analyze clonal acinar development

3D culture systems are a valuable tool for modeling morphogenesis and carcinogenesis of epithelial tissue in a structurally appropriate context. We present a novel approach for 3D cell culture based on a flow-focusing microfluidic system that encapsulates epithelial cells in Matrigel beads. As a model we use prostatic and breast cells and assay for development of acini, polarized cellular spheres enclosing lumen. Each individual bead on average acts as a single 3D cell culture compartment generating one acinus per bead. Compared to standard protocols microfluidics provides increased control over the environment leading to more a uniform acini population. The increased facility of bead manipulation allowed us to isolate single cells which are self-sufficient to fully develop into acini in presence of Matrigel. Furthermore, combination of our microfluidic approach with large particle FACS opens new avenues in high throughput screening on single acini or spheroids.

Abstract 2991: Triple combination of bevacizumab, anti-DLL4 (delta like ligand 4) and trebananib gives enhanced therapeutic effects in three xenograft tumor models

Abstract Bevacizumab is a monoclonal antibody targeting VEGF that is clinically approved for the treatment of multiple indications in oncology, including colorectal, lung and renal cancer as well as glioblastoma. Clinical activity has also been demonstrated in breast and ovarian cancer as well, however, the time periods of benefit for all of these indications is in months (1-5). Multiple pathways are known to participate in the process of angiogenesis, and the limited effects of bevacizumab may represent the maximum activity of targeting just one of them. The purpose of the present study was to examine the effect of simultaneously targeting multiple angiogenic axes including angiopoietins 1 and 2, Dll4 and VEGF. Trebananib is a recombinant peptide-Fc fusion protein that selectively blocks the Tie2 interactions of both Ang1 and Ang2. Trebananib, as well as a recombinant antibody to Dll4 were produced at Amgen, Inc., Thousand Oaks, CA. Bevacizumab was purchased from a local pharmacy. These reagents were employed in the combination treatment of three different preclinical murine tumor models (U87, glioblastoma; MiaPaca and BxPC3, pancreatic carcinoma), one of which (BxPC3) was specifically selected for its relative resistance to VEGF/KDR inhibition. Harlan Athymic nu/nu female mice at 6-7 weeks of age were injected subcutaneously on the right flank with 5 x 106 tumor cells in the presence of Matrigel and treated when the tumors were approximately 300 mm3. The results, as supported by histomorphometric analysis (percent blood vessel area, viable tumor burden), demonstrate that under conditions of optimal biological dosing, the addition of trebananib to a two-way combination of bevacizumab and anti-Dll4 resulted in a statistically significant enhancement of therapeutic effect for the three-way combination, an observation made consistently over 3 different tumor models and 3 different dose levels of bevacizumab and anti-Dll4 (p = or &amp;lt; 0.0024; RMANOVA). Furthermore, the therapeutic effect in all three models approximated prolonged stasis, the ultimate theoretical goal of antiangiogenesis in the treatment of cancer. 1. NEJM, vol. 350, p. 2335, 2004. 2. NEJM, vol. 355, p. 2542, 2006. 3. Lancet, vol. 370, p. 2103, 2007. 4. NEJM, vol. 357, p. 2666, 2007. 5. J. Clin. Oncol., vol. 30, p. 2039, 2012. Citation Format: Bethany Mattson, Jodi Moriguchi, H. Toni Jun, Angela Coxon, Dave Cordover, Steve Kaufman, Jon Oliner, Charlie Starnes. Triple combination of bevacizumab, anti-DLL4 (delta like ligand 4) and trebananib gives enhanced therapeutic effects in three xenograft tumor models. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2991. doi:10.1158/1538-7445.AM2014-2991

Human umbilical cord mesenchymal stromal cells in regenerative medicine

Cells of the human umbilical cord offer tremendous potential for improving human health. Cells from the Wharton’s jelly (umbilical cord stroma) in particular, referred to as human umbilical cord mesenchymal stromal cells (HUCMSCs), hold several advantages that make them appealing for translational research. In the previous issue of Stem Cell Research & Therapy, Chon and colleagues made an important contribution to the HUCMSC literature not only by presenting HUCMSCs as an emerging cell source for intervertebral disc regeneration in general and the nucleus pulposus in particular, but also by demonstrating that an extracellular matrix-based strategy might be preferred over the use of growth factors. By culturing HUCMSCs under hypoxia in serum-free conditions in the presence of Matrigel with laminin-111, they were able to achieve intense collagen II staining by 21 days without the addition of exogenous growth factors. There is tremendous translational significance here in that such raw materials may alleviate the need for the use of growth factors in some instances, and this may have important ramifications in reducing product cost and streamlining regulatory approval. Chon and colleagues provide a promising example of the potential of HUCMSCs, demonstrating the ability to guide HUCMSC differentiation even in the absence of serum and growth factors and supporting the use of HUCMSCs as a viable alternative in intervertebral disc regeneration.

Colony-forming cells in the adult mouse pancreas are expandable in Matrigel and form endocrine/acinar colonies in laminin hydrogel

The study of hematopoietic colony-forming units using semisolid culture media has greatly advanced the knowledge of hematopoiesis. Here we report that similar methods can be used to study pancreatic colony-forming units. We have developed two pancreatic colony assays that enable quantitative and functional analyses of progenitor-like cells isolated from dissociated adult (2-4 mo old) murine pancreas. We find that a methylcellulose-based semisolid medium containing Matrigel allows growth of duct-like "Ring/Dense" colonies from a rare (∼1%) population of total pancreatic single cells. With the addition of roof plate-specific spondin 1, a wingless-int agonist, Ring/Dense colony-forming cells can be expanded more than 100,000-fold when serially dissociated and replated in the presence of Matrigel. When cells grown in Matrigel are then transferred to a Matrigel-free semisolid medium with a unique laminin-based hydrogel, some cells grow and differentiate into another type of colony, which we name "Endocrine/Acinar." These Endocrine/Acinar colonies are comprised mostly of endocrine- and acinar-like cells, as ascertained by RNA expression analysis, immunohistochemistry, and electron microscopy. Most Endocrine/Acinar colonies contain beta-like cells that secrete insulin/C-peptide in response to D-glucose and theophylline. These results demonstrate robust self-renewal and differentiation of adult Ring/Dense colony-forming units in vitro and suggest an approach to producing beta-like cells for cell replacement of type 1 diabetes. The methods described, which include microfluidic expression analysis of single cells and colonies, should also advance study of pancreas development and pancreatic progenitor cells.

Genetic Modification and Recombination of Salivary Gland Organ Cultures

Branching morphogenesis occurs during the development of many organs, and the embryonic mouse submandibular gland (SMG) is a classical model for the study of branching morphogenesis. In the developing SMG, this process involves iterative steps of epithelial bud and duct formation, to ultimately give rise to a complex branched network of acini and ducts, which serve to produce and modify/transport the saliva, respectively, into the oral cavity. The epithelial-associated basement membrane and aspects of the mesenchymal compartment, including the mesenchyme cells, growth factors and the extracellular matrix, produced by these cells, are critical to the branching mechanism, although how the cellular and molecular events are coordinated remains poorly understood. The study of the molecular mechanisms driving epithelial morphogenesis advances our understanding of developmental mechanisms and provides insight into possible regenerative medicine approaches. Such studies have been hampered due to the lack of effective methods for genetic manipulation of the salivary epithelium. Currently, adenoviral transduction represents the most effective method for targeting epithelial cells in adult glands in vivo. However, in embryonic explants, dense mesenchyme and the basement membrane surrounding the epithelial cells impedes viral access to the epithelial cells. If the mesenchyme is removed, the epithelium can be transfected using adenoviruses, and epithelial rudiments can resume branching morphogenesis in the presence of Matrigel or laminin-111. Mesenchyme-free epithelial rudiment growth also requires additional supplementation with soluble growth factors and does not fully recapitulate branching morphogenesis as it occurs in intact glands. Here we describe a technique which facilitates adenoviral transduction of epithelial cells and culture of the transfected epithelium with associated mesenchyme. Following microdissection of the embryonic SMGs, removal of the mesenchyme, and viral infection of the epithelium with a GFP-containing adenovirus, we show that the epithelium spontaneously recombines with uninfected mesenchyme, recapitulating intact SMG glandular structure and branching morphogenesis. The genetically modified epithelial cell population can be easily monitored using standard fluorescence microscopy methods, if fluorescently-tagged adenoviral constructs are used. The tissue recombination method described here is currently the most effective and accessible method for transfection of epithelial cells with a wild-type or mutant vector within a complex 3D tissue construct that does not require generation of transgenic animals.

Human marrow-isolated adult multilineage-inducible (MIAMI) cells protect against peripheral vascular ischemia in a mouse model

The treatment of peripheral vascular disease (PVD) with stem cells potentially offers a promising strategy. We tested marrow-isolated adult multilineage-inducible (MIAMI) cells to induce neovascularization in a mouse model of critical hindlimb ischemia (CLI). CLI was induced in the right hindlimb of Balb/C mice. One million MIAMI cells, normally grown at 3% O₂, were injected in the adductor muscle along the ischemic region. All animals (n = 11 per group) were immunosuppressed with cyclosporine daily for the entire period. Human foreskin fibroblast (HFF) cells and phosphate-buffered saline (PBS) were used as controls. Blood perfusion in the ischemic right and non-ischemic left hindlimbs was measured. Compared with animals receiving HFF cells or PBS, MIAMI cells significantly improved blood perfusion, necrosis and inflammation in the ischemic limb. A fraction of injected MIAMI cells expressed CD31 and von Willebrand factor (vWF). MIAMI cells in vitro, under pro-angiogenic growth conditions, differentiated into endothelial-like cells and expressed endothelial markers such as CD31 and vWF, determined by quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR), and CD31 and kinase insert domain receptor (KDR), determined by immunofluorescence. Moreover, MIAMI cells formed vascular endothelial-like tubules in the presence of matrigel. Bioplex immunoassay analysis showed increased secretion of angiogenic/anti-inflammatory factors by the MIAMI cells under 3% O₂ compared with 21% O₂, including monocyte chemoattractant protein-1 (MCP-1), fractalkine (Ftk), growth-related oncogene (GRO), vascular endothelial growth factor (VEGF), interleukin (IL)-6 and IL-8. Furthermore, transcripts for anti-inflammatory molecules stanniocalcin-1 (STC-1) and tumor necrosis factor-α-stimulated gene 6 (TSG-6) were up-regulated several fold. MIAMI cells can be very useful for patients affected by CLI. MIAMI cells promote blood vessel formation and reduce inflammation and necrosis in ischemic tissue.

Teratoma Formation by Human Embryonic Stem Cells Is Site Dependent and Enhanced by the Presence of Matrigel

When implanted into immunodeficient mice, human embryonic stem cells (hESCs) give rise to teratoma, tumor-like formations containing tissues belonging to all three germ layers. The ability to form teratoma is a sine qua non characteristic of pluripotent stem cells. However, limited data are available regarding the effects of implantation site and the methods employed for implantation on the success rate of teratoma formation. In this study, the rate of teratoma formation in immunodeficient mice was site dependent: subcutaneous (25-100%), intratesticular (60%), intramuscular (12.5%), and under the kidney capsule (100%). Co-injecting the hESCs with Matrigel increased subcutaneous teratoma formation efficiency from 25-40% to 80-100%. We did not observe site-specific differences in the teratoma composition at the histological level. However, subcutaneous teratomas were quite distinct, easy to remove, and caused minimal discomfort to the mice. Also, subcutaneous teratomas displayed larger proportion of solid tissues as opposed to cyst formation that dominated the teratomas formed at the other sites. Interestingly, a chromosomally abnormal hESCs with trisomy 20 formed teratomas where the ratio of differentiated to undifferentiated tissues was significantly decreased suggesting defective pluripotency of the cells. In conclusion, subcutaneous implantation of hESCs in presence of Matrigel appears to be the most efficient, reproducible, and the easiest approach for teratoma formation by hESCs. Also, teratoma formation can be employed to study the development defects exhibited by the chromosomally abnormal hESC lines.

Cell-Polarity Dynamics Controls the Mechanism of Lumen Formation in Epithelial Morphogenesis

Many organs consist of tubes of epithelial cells enclosing a central lumen. How the space of this lumen is generated is a key question in morphogenesis. Two predominant mechanisms of de novo lumen formation have been observed: hollowing and cavitation. In hollowing, the lumen is formed by exocytosis and membrane separation, whereas, in cavitation, the lumen is generated by apoptosis of cells in the middle of the structure [1, 2]. Using MDCK cells in three-dimensional cultures, we found an inverse correlation between polarization efficiency and apoptosis. When cells were grown in collagen, where cells polarized slowly, apoptosis was needed for lumen formation. However, in the presence of Matrigel, which allowed rapid polarization, lumens formed without apoptosis. If polarization in Matrigel was perturbed by blocking formation of the apical surface by RNAi of Cdc42, lumens formed by apoptosis. In a complementary approach, we plated cells at high density so that aggregates formed with little polarity. These aggregates required apoptosis to form lumens, whereas cells plated at low density formed cysts with rapidly polarizing cells and did not need apoptosis to form lumens. The mechanism of lumen formation in the 3D-MDCK model can shift between hollowing and cavitation, depending on cell polarization.

Role of tumor invasiveness, the extracellular matrix, and chromatin sequestration in the susceptibility of uveal melanoma to herpes simplex virus type 1

To better understand determinants of susceptibility/resistance of uveal melanomas to herpes simplex virus type 1 (HSV-1) oncolytic therapy, uveal melanoma cell lines of low (OCM1a) and of high (M619, MUM2B) invasive potential were infected with HSV-1 either in the presence or absence of a laminin-rich extracellular matrix (Matrigel). OCM1a cultures were destroyed faster by HSV-1 than M619 and MUM2B cultures. In the presence of Matrigel, all melanoma cultures demonstrated delayed destruction by HSV-1 relative to Matrigel-free cultures. As sequestration of chromatin is a characteristic feature of highly invasive uveal melanomas that is further increased by exposure to laminin, we explored whether chromatin sequestration could be reversed by HSV-1 infection. HSV-1 infection induced a global reversal of chromatin sequestration in highly invasive uveal melanoma cells. However, this viral effect was first observed only 2 h following virus infection and required novel protein synthesis from input viral DNA. These findings suggest that tumor invasiveness, the spatial relationship of tumor cells to laminin and chromatin sequestration are determinants of susceptibility/resistance of melanomas to HSV-1 oncolytic therapy. Furthermore, these findings indicate for the first time that HSV-1 infection is associated with global exposure of normally highly sequestered cellular DNA in malignant cells.