Adipose-derived stem cell-secreted factors promote early stage follicle development in a biomimetic matrix.
ABSTRACT: Development of primary follicles in vitro benefits from a three-dimensional matrix that is enriched with paracrine factors secreted from feeder cells and mimics the in vivo environment. In this study, we investigated the role of paracrine signaling from adipose-derived stem cells (ADSCs) in supporting primary follicle development in a biomimetic poly(ethylene glycol) (PEG)-based matrix. Follicles co-cultured with ADSCs and follicles cultured in conditioned medium from ADSCs encapsulated in gels (3D CM) exhibited significantly (p < 0.01 and p = 0.09, respectively) improved survival compared to follicles cultured in conditioned medium collected from ADSCs cultured in flasks (2D CM) and follicles cultured without paracrine support. The gene expression of ADSCs suggested that the stem cells maintained their multipotency in the 3D PEG environment over the culture period, regardless of the presence of the follicles, while under 2D conditions the multipotency markers were downregulated. The differences in cytokine signatures of follicles exposed to 3D and 2D ADSC paracrine factors suggest that early cytokine interactions are key for follicle survival. Taken together, the biomimetic PEG scaffold provides a three-dimensional, in vivo-like environment to induce ADSCs to secrete factors which promote early stage ovarian follicle development and survival.
Project description:BACKGROUND:Premature ovarian insufficiency is a common complication of anticancer treatments in young women and girls. The ovary is a complex, highly regulated reproductive organ, whose proper function is contingent upon the bidirectional endocrine, paracrine, and autocrine signaling. These factors facilitate the development of the follicles, the functional units of the ovary, to progress from the gonadotropin-independent, paracrine-controlled early stage to the gonadotropin-dependent, endocrine-controlled later stage. We hypothesized that the low survival rate of individually cultured early-stage follicles could be improved with co-culture of adipose-derived stem cells (ADSCs) that secrete survival- and growth-promoting factors. MATERIALS AND METHODS:Ovarian follicles ranging from 85 to 115 μm in diameter, from 10- to 12-day-old B6CBAF1 mice were mechanically isolated and co-encapsulated with ADSCs within alginate-based 3D culture system. The follicles were cultured for 14 days, imaged using light microscopy every 2 days, and matured at the end. Follicle media were changed every 2 days and collected for hormone measurements. Follicle diameter, morphology, number of transzonal projections, and survival and maturation rates were recorded. Statistical analyses using one- and two-way ANOVA were performed to compare hormone levels, survival of the follicles and ADSCs, oocyte maturation rates, and follicle growth. RESULTS:The co-encapsulation of the follicles with ADSCs increased follicle survival, ranging from 42.4% for the 86-95 μm to 86.2% for the 106-115-μm follicle size group. Co-culture also improved the follicle growth, the rate of antrum formation and oocyte maturation compared to the follicles cultured alone. The levels of androstenedione, estradiol, and progesterone of co-encapsulated follicles increased progressively with time in culture. CONCLUSIONS:To our knowledge, this is the first report of an in vitro system utilizing mouse adipose-derived stem cells to support the development of the mouse follicles. Our findings suggest that co-encapsulation of ADSCs with early-stage follicles supports follicular development, through secretion of cytokines that promote follicular survival, antrum formation, and meiotic competence. The unique 3D culture system that supports the survival of both cell types has translational implications, as ADSCs could be used as an autologous source for in vitro maturation of early-stage human follicles.
Project description:Contemporary systems for in vitro culture of ovarian follicles do not recapitulate the mechanical heterogeneity in mammalian ovary. Here we report microfluidic generation of biomimetic ovarian microtissue for miniaturized three-dimensional (3D) culture of early secondary preantral follicles by using alginate (harder) and collagen (softer) to fabricate the ovarian cortical and medullary tissues, respectively. This biomimetic configuration greatly facilitates follicle development to antral stage. Moreover, it enables in vitro ovulation of cumulus-oocyte complex (COC) from the antral follicles in the absence of luteinizing hormone (LH) and epidermal growth factor (EGF) that are well accepted to be responsible for ovulation in contemporary literature. These data reveal the crucial role of mechanical heterogeneity in the mammalian ovary in regulating follicle development and ovulation. The biomimetic ovarian microtissue and the microfluidic technology developed in this study are valuable for improving in vitro culture of follicles to preserve fertility and for understanding the mechanism of follicle development and ovulation to facilitate the search of cures to infertility due to ovarian disorders.
Project description:Anti-Müllerian hormone (AMH) plays a key role during ovarian follicular development, with local actions associated with a dynamic secretion profile by growing follicles. While results for AMH effects on antral follicle growth and function are consistent among studies in various species, any effects on preantral follicle development remain controversial. Therefore, experiments were conducted to investigate the direct actions and role of AMH during follicle development at the preantral stage. Macaque-specific short-hairpin RNAs (shRNAs) targeting AMH mRNA were incorporated into adenoviral vectors to decrease AMH gene expression in rhesus macaque follicles. Secondary follicles were isolated from adult macaque ovaries and cultured individually in the ultra-low-attachment dish containing defined medium supplemented with follicle-stimulating hormone and insulin for 5 weeks. Follicles were randomly assigned to treatment groups: (a) control, (b) nontargeting control shRNA-vector, (c) AMH shRNA-vector, (d) AMH shRNA-vector + recombinant human AMH, and (e) recombinant human AMH. Follicle survival and growth were assessed. Culture media were analyzed for steroid hormone and paracrine factor concentrations. For in vivo study, the nontargeting control shRNA-vector and AMH shRNA-vector were injected into macaque ovaries. Ovaries were collected 9 days postinjection for morphology and immunohistochemistry assessment. Decreased AMH expression reduced preantral follicle survival and growth in nonhuman primates. Supplemental AMH treatment in the culture media promoted preantral follicle growth to the small antral stage in vitro with increased steroid hormone and paracrine factor production, as well as oocyte maturation. These data demonstrate that AMH is a critical follicular paracrine/autocrine factor positively impacting preantral follicle survival and growth in primates.
Project description:Various toxicants, drugs and their metabolites carry potential ovarian toxicity. Ovarian follicles, the functional unit of the ovary, are susceptible to this type of damage at all stages of their development. However, despite of the large scale of potential negative impacts, assays that study ovarian toxicity are limited. Exposure of cultured ovarian follicles to toxicants of interest served as an important tool for evaluation of toxic effects for decades. Mouse follicles cultured on the bottom of a culture dish continue to serve an important approach for mechanistic studies. In this paper, we demonstrated the usefulness of a hydrogel based 3-dimensional (3D) mouse ovarian follicle culture as a tool to study ovarian toxicity in a different setup. The 3D in vitro culture, based on fibrin alginate interpenetrating network (FA-IPN), preserves the architecture of the ovarian follicle and physiological structure-function relationship. We applied the novel 3D high-throughput (HTP) in vitro ovarian follicle culture system to study the ovotoxic effects of an anti-cancer drug, Doxorobucin (DXR). The fibrin component in the system is degraded by plasmin and appears as a clear circle around the encapsulated follicle. The degradation area of the follicle is strongly correlated with follicle survival and growth. To analyze fibrin degradation in a high throughput manner, we created a custom MATLAB® code that converts brightfield micrographs of follicles encapsulated in FA-IPN to binary images, followed by image analysis. We did not observe any significant difference between manually processed images to the automated MATLAB® method, thereby confirming that the automated program is suitable to measure fibrin degradation to evaluate follicle health. The cultured follicles were treated with DXR at concentrations ranging from 0.005 nM to 200 nM, corresponding to the therapeutic plasma levels of DXR in patients. Follicles treated with DXR demonstrated decreased survival rate in greater DXR concentrations. We observed partial follicle survival of 35% ± 3% (n = 80) in 0.01nM treatment and 48% ± 2% (n = 92) in 0.005nM, which we identified as the IC50 for secondary follicles. In summary, we established a 3D in vitro ovarian follicle culture system that could be used in an HTP approach to measure toxic effects on ovarian follicles.
Project description:R-spondin proteins are adult stem cell growth factors capable of stimulating gut development by activating LGR4, 5, and 6 receptors to promote Wnt signaling. Although multiple Wnt ligands and cognate Frizzled receptors are expressed in the ovary, their physiological roles are unclear. Based on bioinformatic and in situ hybridization analyses, we demonstrated the exclusive expression of R-spondin2 in oocytes of ovarian follicles. In cultured somatic cells from preantral follicles, R-spondin2 treatment (ED50: 3 ng/ml) synergized with Wnt3a to stimulate Wnt signaling. In cultured ovarian explants from prepubertal mice containing preantral follicles, treatment with R-spondin2, similar to follicle stimulating hormone, promoted the development of primary follicles to the secondary stage. In vivo administration of an R-spondin agonist stimulated the development of primary follicles to the antral stage in both immature mice and gonadotropin releasing hormone antagonist-treated adult mice. Subsequent treatment with gonadotropins allowed the generation of mature oocytes capable of undergoing early embryonic development and successful pregnancy. Furthermore, R-spondin agonist treatment of immune-deficient mice grafted with human cortical fragments stimulated the development of primary follicles to the secondary stage. Thus, oocyte-derived R-spondin2 is a paracrine factor essential for primary follicle development, and R-spondin agonists could provide a new treatment regimen for infertile women with low responses to the traditional gonadotropin therapy.
Project description:Percutaneous coronary intervention for coronary artery disease treatment often results in pathological vascular injury, characterized by P-selectin overexpression. Adipose-derived stem cells (ADSCs) therapeutic efficacy remains elusive due to poor ADSCs targeting and retention in injured vessels. Here, conjugated P-selectin binding peptide (PBP) to polyethylene glycol-conjugated phospholipid derivative (DMPE-PEG) linkers (DMPE-PEG-PBP; DPP) are used to facilitate the modification of PBP onto ADSCs cell surfaces via hydrophobic interactions between DMPE-PEG and the phospholipid bilayer. DPP modification neither has influence on ADSCs proliferation nor apoptosis/paracrine factor gene expression. A total of 5 × 10-6 m DPP-modified ADSCs (DPP-ADSCs) strongly binds to P-selectin-displaying activated platelets and endothelial cells (ECs) in vitro and to wire-injured rat femoral arteries when administered by intra-arterial injection. Targeted binding of ADSCs shields injury sites from platelet and leukocyte adhesion, thereby decreasing inflammation at injury sites. Furthermore, targeted binding of ADSCs recovers injured ECs functionality and reduces platelet-initiated vascular smooth muscle cells (VSMCs) chemotactic migration. Targeted binding of DPP-human ADSCs to balloon-injured human femoral arteries is also demonstrated in ex vivo experiments. Overall, DPP-ADSCs promote vascular repair, inhibit neointimal hyperplasia, increase endothelium functionality, and maintain normal VSMCs alignment, supporting preclinical noninvasive utilization of DPP-ADSCs for vascular injury.
Project description:Gelatin hydrogel crosslinked by microbial transglutaminase (mTG) exhibits excellent performance in cell adhesion, proliferation, and differentiation. We examined the gelation time and gel strength of gelatin/mTG hydrogels in various proportions to investigate their physical properties and tested their degradation performances in vitro. Cell morphology and viability of adipose tissue-derived stromal cells (ADSCs) cultured on the 2D gel surface or in 3D hydrogel encapsulation were evaluated by immunofluorescence staining. Cell proliferation was tested via Alamar Blue assay. To investigate the hydrogel effect on cell differentiation, the cardiac-specific gene expression levelsof Nkx2.5, Myh6, Gja1, and Mef2c in encapsulated ADSCs with or without cardiac induction medium were detected by real-time RT-PCR. Cell release from the encapsulated status and cell migration in a 3D hydrogel model were assessed in vitro. Results show that the gelatin/mTG hydrogels are not cytotoxic and that their mechanical properties are adjustable. Hydrogel degradation is related to gel concentration and the resident cells. Cell growth morphology and proliferative capability in both 2D and 3D cultures were mainly affected by gel concentration. PCR result shows that hydrogel modulus together with induction medium affects the cardiac differentiation of ADSCs. The cell migration experiment and subcutaneous implantation show that the hydrogels are suitable for cell delivery.
Project description:Introduction:Paracrine signals, such as soluble cytokines and extracellular matrix cues, are essential for the survival and development of multicellular ovarian follicles. While it is well established that hydrogel-based culture systems successfully support the growth of late-stage follicles for fertility preservation, growing small, early-stage ovarian follicles still proves to be challenging. We hypothesized that paracrine factors secreted from neighboring follicles may be crucial for improving the survival of early-stage follicles in vitro. Methods:To test our hypothesis, we investigated the bi-directional crosstalk of the paracrine signals, such as cell-secreted cytokines, sex hormones and transcription factors (TFs), in follicles encapsulated and cultured for 12 days in alginate in groups of five (5×) and ten (10×). Results:The differential profiles of TF activity and secretome during folliculogenesis were analyzed using TRanscriptional Activity CEllular aRray (TRACER) and data-driven multivariate modeling approach. The mechano- and oxygen-responsive TFs, NF-?B and HIF1, exhibited a unique upregulation signature in 10× follicles. Consistently, levels of proangiogenic factors, such as VEGF-A and angiopoietin-2, were significantly higher in 10× follicles than those in 5× follicles, reaching 269.77 and 242.82 pg/mL on the last day of culture. The analysis of TRACER and secreted cytokines also revealed critical early interactions between cytokines and TFs, correlating with the observed phenotypical and functional differences between conditions. Conclusions:We identified unique signatures of synergism during successful early-stage ovarian follicle development. These findings bring us closer to understanding of mechanisms underlying the downstream effects of interactions between the extracellular microenvironment and early-stage folliculogenesis in vitro.
Project description:BACKGROUND:Dermal papilla cells (DPCs) play a key role in hair growth among the various cell types in hair follicles. Especially, DPCs determine the fate of hair follicle such as anagen to telogen transition and play a pivotal role in androgenic alopecia (AGA). This study was performed to elucidate the hair growth promoting effects of Polygonum multiflorum extract (PM extract) in cultured human DPCs and its underlying mechanisms. METHODS:The effects of PM extract on cultured DPCs were investigated. Cell viability and mitochondrial activity were measured by CCK-8 and JC-1 analysis, respectively. Western blotting, dot blotting, ELISA analysis, immunocytochemistry and real-time PCR analysis were also performed to elucidate the changes in protein and mRNA levels induced by PM extract. 3D cultured DPC spheroids were constructed for mimicking the in vivo DPs. The hair growth stimulatory effect of PM extract was evaluated using human hair follicle organ culture model. RESULTS:PM extract increased the viability and mitochondrial activity in cultured human DPCs in a dose dependent manner. The expression of Bcl2, an anti-apoptotic protein expressed dominantly in anagen was significantly increased and that of BAD, a pro-apoptotic protein expressed in early catagen was decreased by PM extract in cultured DPCs and/or 3D DPC spheroid culture. PM extract also decreased the expression of catagen inducing protein, Dkk-1. Growth factors including IGFBP2, PDGF and VEGF were increased by PM extract, revealed by dot blot protein analysis. We also have found that PM extract could reverse the androgenic effects of dihydrotestosterone (DHT), the most potent androgen. Finally, PM extract prolonged the anagen of human hair follicles by inhibiting catagen entry in human hair follicle organ culture model. CONCLUSION:Our data strongly suggest that PM extract could promote hair growth by elongating the anagen and/or delaying the catagen induction of hair follicles through activation of DPCs.
Project description:Can cultured follicles model the ovarian cycle, including follicular- and luteal-phase hormone synthesis patterns and ovulation?Under gonadotrophin stimulation, murine follicles grown in an encapsulated three-dimensional system ovulate in vitro and murine and human follicle hormone synthesis mimics follicular and luteal phases expected in vivo.Studies of the human ovary and follicle function are limited by the availability of human tissue and lack of in vitro models. We developed an encapsulated in vitro follicle growth (eIVFG) culture system, which preserves 3D follicular structure. Thus far, the alginate system has supported the culture of follicles from mice, dog, rhesus macaque, baboon and human. These studies have shown that cultured follicles synthesize steroid hormones similar to those observed during the follicular phase in vivo.Cultured murine follicles were treated with human chorionic gonadotrophin (hCG) and epidermal growth factor (EGF) and either assayed for luteinization or removed from alginate beads and assayed for ovulation. Human follicles were also cultured, treated with follicle-stimulating hormone (FSH), hCG and EGF to mimic gonadotrophin changes throughout the ovarian cycle, and culture medium was assayed for hormone production.Murine and human follicles were cultured in alginate hydrogel and hormone production [17?-estradiol, progesterone, inhibin A, inhibin B, activin A and anti-Müllerian hormone (AMH)] was quantified in medium by enzyme-linked immuno assay (ELISA). Human ovarian tissue was acquired from females between 6 and 34 years of age with a cancer diagnosis. These participants were undergoing ovarian tissue cryopreservation at National Physicians Cooperative sites as part of the Oncofertility Consortium.When grown in this system, 96% of mouse follicles ovulated in response to hCG and released meiotically competent eggs. Ovulated follicles recapitulated transcriptional, morphologic and hormone synthesis patterns post-luteinizing hormone (LH/hCG). In addition to rodent follicles, individual human follicles secreted steroid and peptide hormones that mimicked the patterns of serum hormones observed during the menstrual cycle.This was a descriptive study of an in vitro model of ovulation and the ovarian hormone cycle. The ovulation studies were limited to murine tissue and further studies are needed to optimize conditions using other species.The eIVFG system reliably phenocopies the in vivo ovarian cycle and provides a new tool to study human follicle biology and the influence of cycling female hormones on other tissue systems in vitro.This work was supported by NIH U54 HD041857, NIH U54 HD076188, NIH UH2 E5022920, NIH UH3 TR001207 and F30 AG040916 (R.M.S.). The content is solely the responsibility of the authors and does not necessarily represent the official views of the NIH. The funders had no role in study design, data collection and analysis, decision to publish or preparation of the manuscript.