Human embryonic stem cells (hESC) have emerged as attractive candidates for cell-based therapies that are capable of restoring lost cell and tissue function. These unique cells are able to self-renew indefinitely and have the capacity to differentiate in to all three germ layers (ectoderm, endoderm and mesoderm). Harnessing the power of these pluripotent stem cells could potentially offer new therapeutic treatment options for a variety of medical conditions. Since the initial derivation of hESC lines in 1998, tremendous headway has been made in better understanding stem cell biology and culture requirements for maintenance of pluripotency. The approval of the first clinical trials of hESC cells for treatment of spinal cord injury and macular degeneration in 2010 marked the beginning of a new era in regenerative medicine. Yet it was clearly recognized that the clinical utility of hESC transplantation was still limited by several challenges. One of the most immediate issues has been the exposure of stem cells to animal pathogens, during hESC derivation and during in vitro propagation. Initial culture protocols used co-culture with inactivated mouse fibroblast feeder (MEF) or human feeder layers with fetal bovine serum or alternatively serum replacement proteins to support stem cell proliferation. Most hESC lines currently in use have been exposed to animal products, thus carrying the risk of xeno-transmitted infections and immune reaction. This mini review provides a historic perspective on human embryonic stem cell culture and the evolution of new culture models. We highlight the challenges and advances being made towards the development of xeno-free culture systems suitable for therapeutic applications.

Vascular diseases are characterized by the over-proliferation and migration of aortic smooth muscle cells (SMCs), and degradation of extracellular matrix (ECM) within the vessel wall, leading to compromise in cell-cell and cell-matrix signaling pathways. Tissue engineering approaches to regulate SMC over-proliferation and enhance healthy ECM synthesis showed promise, but resulted in low crosslinking efficiency. Here, we report the benefits of exogenous nitric oxide (NO) cues, delivered from S-Nitrosoglutathione (GSNO), to cell proliferation and matrix deposition by adult human aortic SMCs (HA-SMCs) within three-dimensional (3D) biomimetic cocultures. A coculture platform with two adjacent, permeable 3D culture chambers was developed to enable paracrine signaling between vascular cells. HA-SMCs were cultured in these chambers within collagen hydrogels, either alone or in the presence of human aortic endothelial cells (HA-ECs) cocultures, and exogenously supplemented with varying GSNO dosages (0-100 nM) for 21 days. Results showed that EC cocultures stimulated SMC proliferation within GSNO-free cultures. With increasing GSNO concentration, HA-SMC proliferation decreased in the presence or absence of EC cocultures, while HA-EC proliferation increased. GSNO (100 nM) significantly enhanced the protein amounts synthesized by HA-SMCs, in the presence or absence of EC cocultures, while lower dosages (1-10 nM) offered marginal benefits. Multi-fold increases in the synthesis and deposition of elastin, glycosaminoglycans, hyaluronic acid, and lysyl oxidase crosslinking enzyme (LOX) were noted at higher GSNO dosages, and coculturing with ECs significantly furthered these trends. Similar increases in TIMP-1 and MMP-9 levels were noted within cocultures with increasing GSNO dosages. Such increases in matrix synthesis correlated with NO-stimulated increases in endothelial nitric oxide synthase (eNOS) and inducible nitric oxide synthase (iNOS) expression within EC and SMC cultures, respectively. Results attest to the benefits of delivering NO cues to suppress SMC proliferation and promote robust ECM synthesis and deposition by adult human SMCs, with significant applications in tissue engineering, biomaterial scaffold development, and drug delivery.

A major onset of heart failure is myocardial infarction, which causes the myocardium to lose cardiomyocytes and transform into a scar tissue. Since mammalian infarcted cardiac tissue has a limited ability to regenerate, alternative strategies including implantation of tissue-engineered scaffolds at the site of damaged myocardium have been explored. The goal is to enable in situ cardiac reconstruction at the injured myocardium site, replace the lost cardiomyocytes, deliver the required biomolecules, and remodel the extracellular matrix (ECM). ECM synthesis and deposition by cardiomyocytes within such scaffolds remains categorically unexplored. Here, we investigated the survival, ECM synthesis and deposition, and matrix metalloproteinases (MMPs) release by cardiomyocytes within three-dimensional (3D) substrates. Rat cardiomyocytes were cultured for three weeks within two structurally different substrates: 3D collagen hydrogels or polycaprolactone (PCL) nanofibrous scaffolds. The concentration and composition of the hydrogels was varied, while PCL nanofibers were surface-modified with various ECM proteins. Results showed that myocyte attachment and survival was higher within collagen hydrogels, while myocyte alignment and beating was noted only within PCL scaffolds. Total protein synthesis by myocytes within PCL scaffolds was significantly higher compared to that within collagen hydrogels, although more protein was deposited as matrix within hydrogels. Significant ECM synthesis and matrix deposition, TIMP-1, and MMP release were noted within modified collagen hydrogels and PCL nanofiber scaffolds. These results were qualitatively confirmed by imaging techniques. Results attest to the prominent role of scaffold composition and architecture in influencing cardiomyocyte phenotype, matrix synthesis and cytokines release, with significant applications in cardiac tissue remodeling strategies.

Background Encapsulation of follicles within a biomatrix is one approach to maintaining 3-D follicle architecture during culture. Hyaluronan is one component of the natural extracellular matrix (ECM) that provides support to cells in vivo. This report describes the application of a novel tyramine-linked hyaluronan for 3-D in vitro follicle culture and the production of developmentally competent metaphase II oocytes. Materials and Methods Enzymatically isolated mouse preantral follicles or follicle clusters (FL-C) from fresh or vitrified ovaries were encapsulated in 3 mg/ml of hyaluronan gel (HA). Follicle growth, antrum formation and meiotic maturation to metaphase II oocytes was monitored. Chromatin staining was used to assess GV oocyte progression towards meiotic competence. Functional competence of in vitro matured (IVM) oocytes was evaluated by in vitro fertilization and ability to develop to blastocyst. Modifying the HA gel by inclusion of laminin (HA-LM), mouse sarcoma extracellular matrix (Matrigel;HA-MG) or placental extracellular matrix (HA-PM) was also tested to see if this might further enhance IVM outcomes. Results A total of 402 preantral follicles were cultured in HA gel. After hCG trigger, 314 oocyte-cumulus complexes ovulated from the embedded follicles. Meiotic maturation rate to the metaphase II stage was 73% (228/314). After insemination 83% (188/228) of IVM oocytes fertilized with a subsequent blastulation rate of 46% (87/188). A pilot transfer study with 3 recipient mice resulted in the birth of a single pup. HA gel supported individually isolated follicles as well ovarian tissue fragments containing clusters of 6–8 preantral follicles. Meiotic maturation was lower with FL-clusters from vitrified versus fresh ovaries (34% and 55%, respectively; p < 0.007). Modification of the HA gel with ECMs or laminin affected antrum formation and follicle retention. Maturation rates to the metaphase II stage were however not significantly different: 74% for HA gel alone as compared to HA-LM (67%), HA-MG (56%) and HA-PM (58%). Conclusion Hyaluronan gel is an effective and versatile extracellular matrix based biomaterial for 3-D culture of ovarian follicles. This culture model allowed ovulation of functionally competent metaphase II oocytes, capable of fertilization, genomic activation and blastulation. Future testing with human follicles that require longer in vitro culture times should be considered.

Background Within the ovary, bidirectional communication between oocytes and surrounding granulosa cells is required for appropriate nuclear and cytoplasmic maturation (1, 10–14). The 3-D architecture of the growing follicle is however difficult to maintain during in vitro maturation using conventional 2-D tissue culture systems. Encapsulation of follicles within a biomatrix is one approach to maintaining 3-D follicle architecture during culture. Hyaluronan is one component of natural extracellular matrices (ECM) that provide support to cells in vivo. This report describes the application of hyaluronan for 3-D in vitro follicle culture and the production of developmentally competent metaphase II oocytes. Materials and Methods A novel tyramine-linked hyaluronan (HA) hydrogel was used for follicle encapsulation. Enzymatically isolated mouse preantral follicles or follicle clusters (FL-C) from fresh or vitrified ovaries were encapsulated in 3 mg/ml HA gel for in vitro follicle maturation. Follicle growth, antrum formation and meiotic maturation to metaphase II oocytes was monitored. Chromatin staining was used to assess GV oocyte progression towards meiotic competence. Spindle morphology was evaluated in metaphase II oocytes using immunostaining and polarized light. Functional competence of in vitro matured oocytes was evaluated by in vitro fertilization and ability to develop to blastocyst. Modifying the HA gel by inclusion of laminin (HA-LM), mouse sarcoma extracellular matrix (Matrigel;HA-MG) or placental extracellular matrix (HA-PM) was also tested to see if this might further enhance IVM outcomes. Results A total of 402 pre-antral follicles were embedded in HA gel. Antrums were observed in 55% of follicles by day 8. Post-hCG trigger, 314 oocyte-cumulus complexes ovulated from the cultured follicles and 84% (264/314) underwent GVBD. The maturation rate to MII was 72.6%. (228/314). The fertilization rate was 82.5% (188/228). The subsequent blastulation rate with IVM oocytes was 46.3% (87/188). Blastocysts were vitrified for later use. Thawed blastocysts were transferred into three recipient and resulted in the birth of a single pup. HA gel supported individually isolated follicles as well ovarian tissue fragments containing clusters of 6–8 preantral follicles. Meiotic maturation to the metaphase II stage was significantly lower with FL-clusters from vitrified versus fresh ovaries (34% and 55%, respectively; p < 0.007). Modification of the HA gel with ECMs or laminin affected antrum formation and follicle retention within the gel during in vitro culture. Significantly lower antrum formation was noted amongst follicles in HA-LM (29%), HA-MG (18%) and HA-PM (26%) as compared to HA alone, 48%; p = 0.006). The HA-Matrigel encapsulated follicle treatment group had a lower rate of ovulation (37%) as compared to all three of the other treatment groups ( 65–67%; P = 0.001). The rate of maturation to metaphase II oocytes was 74% for HA gel alone as compared to HA-LM (67%), HA-MG (56%) and HA-PM (58%) but these differences were not statistically significant. HA alone proved to be entirely sufficient for supporting the development of mature competent metaphase II oocytes. Conclusion Tyramine-linked hyaluronan gel is an effective ECM based biomaterial for 3-D culture of ovarian follicles in clusters or individually. In vitro matured metaphase II oocytes are functionally competent, capable of being fertilized and developing to the blastocyst stage. The versatility, simplicity of use and ability to be used at various rigidities give this new biomatrix numerous advantages, making it particularly attractive for ovarian follicle culture.

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for 6hrs and RNA-seq was performed. Significant differentially expressed genes (SDEGs) and upregulated Gene Ontology (GO) cellular pathways were identified. To determine if gene expression changes were associated with changes in cellular response, transwell assays were performed to assess HTR-8 cell migration and invasion after exposure to 2.5% O 2 or 21% O 2 for 6hrs and 24hrs. For transwell migration assays (n¼6), number of migrated cells through culture inserts were counted. For invasion assays (n¼6), number of cells that invaded into Matrigel and collagen I matrices were counted. Means were compared using unpaired t tests and statistical significance was defined as p <0.05. To determine if changes in gene expression induced by exposure of HTR-8 to 2.5% O 2 were present in 2 nd trimester CVS from PE pregnancies relative to healthy pregnancies, CVS were collected and analyzed under an IRB approved protocol. RNA-seq of CVS from PE (n¼2) and healthy (n¼4) pregnancies was performed and DEGs were identified.RESULTS: GO analysis showed that exposure of HTR-8 to 2.5% O 2 for 6hrs upregulated cellular migration pathways. SDEGs included genes involved in TB migration and invasion (MMP9, TIMP1, and PAPPA), as well as genes involved in tumor migration and invasion (ACTA2, MFAP4, SNAI2, SLCO4A1, GDF15, KLF5, ZBTB20, and ZNF703). HTR-8 cell migration and invasion, through both collagen I and Matrigel matrices, was significantly increased after exposure to 2.5% O 2 for 24hrs but not 6hrs. Analysis of RNA-seq data from CVS of PE and healthy pregnancies identified 17 significant DEGs, 4 of which (FAT2, SPON2, RASGRF2, and SCLO4A1) were decreased in CVS from PE pregnancies and are involved in cellular migration and invasion.CONCLUSIONS: Exposure of 1 st trimester TBs to physiologic hypoxic conditions induces expression of genes associated with cellular migration and invasion and increases 1 st trimester TB migration and invasion in vitro. Decreased expression of migration and invasion genes in CVS from PE pregnancies may impair TB migration and invasion in the 2 nd trimester of pregnancy, resulting in inadequate spiral artery transformation and prolonged hypoxia, preceding the development of PE.

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