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Promoting extracellular matrix remodeling via ascorbic acid enhances the survival of primary ovarian follicles encapsulated in alginate hydrogels.

ABSTRACT: The in vitro growth of ovarian follicles is an emerging technology for fertility preservation. Various strategies support the culture of secondary and multilayer follicles from various species including mice, non-human primate, and human; however, the culture of early stage (primary and primordial) follicles, which are more abundant in the ovary and survive cryopreservation, has been limited. Hydrogel-encapsulating follicle culture systems that employed feeder cells, such as mouse embryonic fibroblasts (MEFs), stimulated the growth of primary follicles (70-80 µm); yet, survival was low and smaller follicles (<70 µm) rapidly lost structure and degenerated. These morphologic changes were associated with a breakdown of the follicular basement membrane; hence, this study investigated ascorbic acid based on its role in extracellular matrix (ECM) deposition/remodeling for other applications. The selection of ascorbic acid was further supported by a microarray analysis that suggested a decrease in mRNA levels of enzymes within the ascorbate pathway between primordial, primary, and secondary follicles. The supplementation of ascorbic acid (50 µg/mL) significantly enhanced the survival of primary follicles (<80 µm) cultured in alginate hydrogels, which coincided with improved structural integrity. Follicles developed antral cavities and increased to diameters exceeding 250 µm. Consistent with improved structural integrity, the gene/protein expression of ECM and cell adhesion molecules was significantly changed. This research supports the notion that modifying the culture environment (medium components) can substantially enhance the survival and growth of early stage follicles.

SUBMITTER: Tagler D

PROVIDER: S-EPMC4232184 | biostudies-literature | 2014 Jul

REPOSITORIES: biostudies-literature

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Promoting extracellular matrix remodeling via ascorbic acid enhances the survival of primary ovarian follicles encapsulated in alginate hydrogels.

Tagler David D Makanji Yogeshwar Y Tu Tao T Bernabé Beatriz Peñalver BP Lee Raymond R Zhu Jie J Kniazeva Ekaterina E Hornick Jessica E JE Woodruff Teresa K TK Shea Lonnie D LD

Biotechnology and bioengineering 20140122 7

The in vitro growth of ovarian follicles is an emerging technology for fertility preservation. Various strategies support the culture of secondary and multilayer follicles from various species including mice, non-human primate, and human; however, the culture of early stage (primary and primordial) follicles, which are more abundant in the ovary and survive cryopreservation, has been limited. Hydrogel-encapsulating follicle culture systems that employed feeder cells, such as mouse embryonic fibr ...[more]

PMID: 24375265

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Project description:Hydrogel-encapsulating culture systems for ovarian follicles support the in vitro growth of secondary follicles from various species including mouse, non-primate human, and human; however, the growth of early stage follicles (primary and primordial) has been limited. While encapsulation maintains the structure of early stage follicles, feeder cell populations, such as mouse embryonic fibroblasts (MEFs), are required to stimulate growth and development. Hence, in this report, we investigated feeder-free culture environments for early stage follicle development. Mouse ovarian follicles were encapsulated within alginate hydrogels and cultured in various growth medium formulations. Initial studies employed embryonic stem cell medium formulations as a tool to identify factors that influence the survival, growth, and meiotic competence of early stage follicles. The medium formulation that maximized survival and growth was identified as ?MEM/F12 supplemented with fetuin, insulin, transferrin, selenium, and follicle stimulating hormone (FSH). This medium stimulated the growth of late primary (average initial diameter of 80 µm) and early secondary (average initial diameter of 90 µm) follicles, which developed antral cavities and increased to terminal diameters exceeding 300 µm in 14 days. Survival ranged from 18% for 80 µm follicles to 36% for 90 µm follicles. Furthermore, 80% of the oocytes from surviving follicles with an initial diameter of 90-100 µm underwent germinal vesicle breakdown (GVBD), and the percentage of metaphase II (MII) eggs was 50%. Follicle/oocyte growth and GVBD/MII rates were not significantly different from MEF co-culture. Survival was reduced relative to MEF co-culture, yet substantially increased relative to the control medium that had been previously used for secondary follicles. Continued development of culture medium could enable mechanistic studies of early stage folliculogenesis and emerging strategies for fertility preservation.

Project description:Myogenic differentiation, cell fusion, and myotube formation of skeletal muscle progenitor cells (SMPCs) have key roles during skeletal muscle development and repair. However, after isolation from living tissue and transition to culture dishes, SMPCs gradually lose their function and stop propagating due to the absence of extracellular matrix (ECM). Despite encouraging results of experiments using ECM components in cell culture for maintenance and propagation of some tissue types, the benefits of this approach on SMPC culture are limited, because the bioactive molecules and proteins instantly release and are degraded during culture. In this study, we developed a novel approach to enhance the proliferation and differentiation of human skeletal muscle progenitor cells (hSMPCs) in vitro with skeletal muscle ECM in combination with a modified alginate hydrogel conjugated with gelatin and heparin (Alg-G-H) as a substrate. This Alg-G-H substrate, together with skeletal muscle ECM, significantly enhanced cell expansion, differentiation, and maturation of hSMPCs compared with individual substrata (i.e. gelatin, Matrigel®, or ECM alone). In Western-blot and immunocytochemical analyses, the Alg-G-H-ECM predominantly enhanced expression of skeletal myogenesis markers (MyoD, Myf5, Myogenin, Desmin and Myosin) and myotube formation in hSMPCs. This study demonstrated that combining Alg-G-H substrates with skeletal muscle ECM modulated homeostasis of cell proliferation, differentiation, and maturation of hSMPCs by releasing signaling molecules and growth factors. This technique could be a cost-effective tool for in vitro skeletal muscle cell differentiation and maturation, with potential applications in tissue regeneration and drug development.Statement of significanceAlginate based biomaterials are commonly used in tissue engineering and regenerative medicine field, however, the inefficient sequestration of growth factors restricted its utilization. In this study, a novel alginate based substrates was produced covalently modified with gelatin and heparin, in order to capture more effective cytokines and proteins in the culture milieu, keep homeostasis for cell survival and tissue regeneration with growth factor sequestration and long-term release capacities. Combining with skeletal muscle derived ECM, the modified Alginate-Gelatin-Heparin gel could most effectively mimic the tissue specific microenvironment to support skeletal muscle progenitor cells proliferation, differentiation and myotube formation. Additionally, the economical and practical features will make it more promising in high-throughput application for regenerative medicine research.

Project description:BackgroundHuman amniotic epithelial cells (hAECs) exhibit a strong capability to restore ovarian function in chemotherapy-induced premature ovarian failure (POF). However, the therapeutic efficacy of hAECs is usually affected by the limited number and proliferative ability of grafted hAECs in target organs. The transplantation of stem cells encapsulated in sodium alginate-bioglass (SA-BG) composite hydrogel has recently been shown to be an effective strategy for tissue regeneration. The current study aims to investigate the therapeutic potential of hAECs or hAEC-derived conditioned medium (CM) encapsulated in SA-BG in mice with chemotherapy-induced POF.MethodsC57BL/6 mice were intraperitoneally injected with chemotherapy drugs to induce POF. hAECs or CM were harvested and encapsulated in SA-BG composite hydrogel, which were transplanted onto the injured ovaries of mice with POF. Follicle development, granulosa cell function, and ovarian angiogenesis were evaluated by morphological methods. To further elucidate the effect of SA-BG-encapsulated hAECs/CM on vascularization, the tube formation of human umbilical vein epithelial cells (hUVECs) was conducted in vitro. Cytokine array and ELISA were used to analyze and quantify the effects of bioactive components released by SA-BG on the secretion of angiogenic factors by hAECs.ResultsThe transplantation of SA-BG-encapsulated hAECs/CM restored follicle development, repaired granulosa cell function, and enhanced ovarian angiogenesis in POF mice. The further study showed that SA-BG significantly promoted the tube formation of hUVECs in vitro. Moreover, encapsulating hAECs could facilitate the effect of SA-BG on inducing the formation of the capillary tube in a paracrine manner. In addition, we found that SA-BG extracts significantly enhanced the viability of hAECs and stimulated the secretion of pro-angiogenic factors of hAECs. Notably, compared with SA-BG/CM, SA-BG/hAECs achieve better therapeutic effects, possibly because stimulation of BG enhanced the viability and paracrine capacity of hAECs.ConclusionsThe present study initially demonstrates that SA-BG-encapsulated hAECs or CM can exert a therapeutic effect on chemotherapy-induced POF mainly by protecting granulosa cell function and enhancing ovarian vascularization, which might provide a novel strategy for the delivery of hAECs for treating POF.

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Promoting extracellular matrix remodeling via ascorbic acid enhances the survival of primary ovarian follicles encapsulated in alginate hydrogels.

Publications

Promoting extracellular matrix remodeling via ascorbic acid enhances the survival of primary ovarian follicles encapsulated in alginate hydrogels.

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