Adipose-derived stem cell-secreted factors promote early stage follicle development in a biomimetic matrix.
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ABSTRACT:
SUBMITTER: Tomaszewski CE
PROVIDER: S-EPMC6351215 | biostudies-literature | 2019 Jan
REPOSITORIES: biostudies-literature
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Project description:BackgroundPremature 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 methodsOvarian 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.ResultsThe 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.ConclusionsTo 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.
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Project description:Scar formation is an intractable medical problem that appears after skin wounds have healed. Recent research has shown that exosomes secreted by human adipose mesenchymal stem cells (ASC-Exos) can benefit wound healing. To further explore the therapeutic potential of ASC-Exos, we investigated their effects on mitigating scar formation, and the underlying mechanisms of these effects. We found that intravenous injection of ASC-Exos decreased the size of scars and increased the ratio of collagen III to collagen I in murine incisional wounds. Exosome treatment also prevented the differentiation of fibroblasts into myofibroblasts and increased the ratio of transforming growth factor-?3 (TGF-?3) to TGF-?1 in vivo. Additionally, we found that ASC-Exos increased the matrix metalloproteinases-3 (MMP3) expression of skin dermal fibroblasts by activating the ERK/MAPK pathway, leading to a high ratio of MMP3 to tissue inhibitor of matrix metalloproteinases-1 (TIMP1), which is also beneficial for the remodelling of extracellular matrix (ECM). In conclusion, our results demonstrated that ASC-Exos promote ECM reconstruction in cutaneous wound repair by regulating the ratios of collagen type III: type I, TGF-?3:TGF-?1 and MMP3:TIMP1, and by regulating fibroblast differentiation to mitigate scar formation. Therefore, the application of ASC-Exos may be a novel therapeutic approach for scarless wound repair.
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Project description:Epithelial ovarian cancer (EOC) patients frequently develop peritoneal metastasis, especially in the human omentum. However, the mechanism underlying this propensity remains unknown. A previous study found that human omental adipose-derived mesenchymal stem cells are potentially involved in ovarian cancer growth and metastasis, but the results were inconsistent and even contradictory. In addition, the underlying mechanisms of visceral adipose metastasis remain poorly understood. Here, our goal is to clarify the role and mechanism of human omental adipose-derived mesenchymal stem cells (HO-ADSCs) in EOC cancer growth and metastasis. We first found that human omental tissue conditioned medium (HO-CM) enhances EOC cell function. Subsequent coculture studies indicated that HO-ADSCs increase the growth, migratory and invasive capabilities of ovarian cancer cells. Then, we demonstrated that exosomes secreted by HO-ADSCs (HO-ADSC exosomes) enhanced ovarian cancer cell function, and further mechanistic studies showed that the FOXM1, Cyclin F, KIF20A, and MAPK signaling pathways were involved in this process. In addition, subcutaneous tumorigenesis and peritoneal metastatic xenograft experiments provided evidence that HO-ADSC exosomes promote ovarian cancer growth and metastasis in vivo. Finally, our clinical studies provided evidence that ascites from ovarian cancer patients enhance EOC cell line proliferation, migration, and invasion in vitro. The present study indicated that HO-ADSC exosomes are secreted into ascites and exert a tumor-promoting effect on EOC growth and metastasis, providing a new perspective and method to develop future novel therapeutic strategies for the treatment of ovarian cancer.
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Project description:Fat grafting is an established clinical intervention to promote tissue repair. The role of the fat's extracellular matrix (ECM) in regeneration is largely neglected. We investigated in vitro the use of human adipose tissue-derived ECM hydrogels as release platform for factors secreted by adipose-derived stromal cells (ASCs). Lipoaspirates from nondiabetic and diabetic donors were decellularized. Finely powdered acellular ECM was evaluated for cell remainders and DNA content. Acellular ECM was digested, and hydrogels were formed at 37°C and their viscoelastic relaxation properties investigated. Release of ASC-released factors from hydrogels was immune assessed, and bio-activity was determined by fibroblast proliferation and migration and endothelial angiogenesis. Acellular ECM contained no detectable cell remainders and negligible DNA contents. Viscoelastic relaxation measurements yielded no data for diabetic-derived hydrogels due to gel instability. Hydrogels released several ASC-released factors concurrently in a sustained fashion. Functionally, released factors stimulated fibroblast proliferation and migration as well as angiogenesis. No difference between nondiabetic and diabetic hydrogels in release of factors was measured. Adipose ECM hydrogels incubated with released factors by ASC are a promising new therapeutic modality to promote several important wound healing-related processes by releasing factors in a controlled way.
| S-EPMC6593768 | biostudies-literature
Project description:Adipose-derived stem cells (ASCs) have shown efficacy in promoting hair growth, while DKK1 inhibits the WNT pathway, which is associated with hair loss. Our study focused on investigating the expression of DKK1 in alopecia areata (AA), a condition characterised by significant increases in the DKK1 levels in human and mouse ASCs. Treatment of interferon-γ increased the expression of DKK1 via STAT3 phosphorylation in ASCs. Treatment with recombinant DKK1 resulted in a decrease of cell growth in outer root sheath cells, whereas the use of a DKK1 neutralising antibody promoted hair growth. These results indicate that ASCs secrete DKK1, playing a crucial role in the progression and development of AA. Consequently, we generated DKK1 knockout (KO) ASCs using the Crispr/Cas9 system and evaluated their hair growth-promoting effects in an AA model. The DKK1 KO in ASCs led to enhanced cell motility and reduced cellular senescence by activating the WNT signalling pathway, while it reduced the expression of inflammatory cytokines by inactivating the NF-kB pathway. As expected, the intravenous injection of DKK1-KO-ASCs in AA mice, and the treatment with a conditioned medium derived from DKK1-KO-ASCs in hair organ culture proved to be more effective compared with the use of naïve ASCs and their conditioned medium. Overall, these findings suggest that DKK1 represents a novel therapeutic target for treating AA, and cell therapy using DKK1-KO-ASCs demonstrates greater efficiency.
| S-EPMC10905327 | biostudies-literature
Project description:Extracellular vesicles (EVs) such as microvesicles (MIVs) play an important role in intercellular communications. MIVs are small membrane vesicles sized 100-1000 nm in diameter that are released by many types of cells, such as mesenchymal stem cells (MSCs), tumor cells and adipose-derived stem cells (ADSC). As EVs can carry out autocrine and paracrine functions by controlling multiple cell processes, it is conceivable that EVs can be used as delivery vehicles for treating several clinical conditions, such as to improve cardiac angiogenesis after myocardial infarction (MI). Here, we seek to investigate whether ADSC-derived MIVs contain microRNAs that regulate angiogenesis and affect cell migration of endothelial cells. We first characterized the ADSC-derived MIVs and found that the MIVs had a size range of 100-300 nm, and expressed the MIV marker protein Alix. We then analyzed the microRNAs in ADSCs and ADSC-derived MIVs and demonstrated that ADSC-derived MIVs selectively released a panel of microRNAs, several of which were related to angiogenesis, including two members of the let-7 family. Furthermore, we demonstrated that ADSC-derived MIVs promoted the cell migration and invasion of the HUVEC endothelial cells. The PKH26-labeled ADSC-derived MIVs were effectively uptaken into the cytoplasm of HUVEC cells. Collectively, our results demonstrate that the ADSC-derived MIVs can promote migration and invasion abilities of endothelial cells, suggesting pro-angiogenetic potential. Future studies should focus on investigating the roles and mechanisms through which ADSC-derived MIVs regulate angiogenesis.
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Project description:Craniomaxillofacial (CMF) reconstruction is a challenging clinical dilemma. It often necessitates skin replacement in the form of autologous graft or flap surgery, which differ from one another based on hypodermal/dermal content. Unfortunately, both approaches are plagued by scarring, poor cosmesis, inadequate restoration of native anatomy and hair, alopecia, donor site morbidity, and potential for failure. Therefore, new reconstructive approaches are warranted, and tissue engineered skin represents an exciting alternative. In this study, we demonstrated the reconstruction of CMF full-thickness skin defects using intraoperative bioprinting (IOB), which enabled the repair of defects via direct bioprinting of multiple layers of skin on immunodeficient rats in a surgical setting. Using a newly formulated patient-sourced allogenic bioink consisting of both human adipose-derived extracellular matrix (adECM) and stem cells (ADSCs), skin loss was reconstructed by precise deposition of the hypodermal and dermal components under three different sets of animal studies. adECM, even at a very low concentration such as 2 % or less, has shown to be bioprintable via droplet-based bioprinting and exhibited de novo adipogenic capabilities both in vitro and in vivo. Our findings demonstrate that the combinatorial delivery of adECM and ADSCs facilitated the reconstruction of three full-thickness skin defects, accomplishing near-complete wound closure within two weeks. More importantly, both hypodermal adipogenesis and downgrowth of hair follicle-like structures were achieved in this two-week time frame. Our approach illustrates the translational potential of using human-derived materials and IOB technologies for full-thickness skin loss.
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Project description:Repair of soft tissue defects resulting from lumpectomy or mastectomy has become an important rehabilitation process for breast cancer patients. This study aimed to provide an adipose tissue engineering platform for soft tissue defect repair by combining decellularized human adipose tissue extracellular matrix (hDAM) and human adipose-derived stem cells (hASCs). To derive hDAM incised human adipose tissues underwent a decellularization process. Effective cell removal and lipid removal were proved by immunohistochemical analysis and DNA quantification. Scanning electron microscopic examination showed a three-dimensional nanofibrous architecture in hDAM. The hDAM included collagen, sulfated glycosaminoglycan, and vascular endothelial growth factor, but lacked major histocompatibility complex antigen I. hASC viability and proliferation on hDAM were proven in vitro. hDAM implanted subcutaneously in Fischer rats did not cause an immunogenic response, and it underwent remodeling, as indicated by host cell infiltration, neovascularization, and adipose tissue formation. Fresh fat grafts (Coleman technique) and engineered fat grafts (hDAM combined with hASCs) were implanted subcutaneously in nude rats. The implanted engineered fat grafts maintained their volume for 8 weeks, and the hASCs contributed to adipose tissue formation. In summary, the combination of hDAM and hASCs provides not only a clinically translatable platform for adipose tissue engineering, but also a vehicle for elucidating fat grafting mechanisms.
| S-EPMC3965366 | biostudies-literature
Project description:Human adipose stem cells (hASCs) can differentiate into a variety of phenotypes. Native extracellular matrix (e.g., demineralized bone matrix or small intestinal submucosa) can influence the growth and differentiation of stem cells. The hypothesis of this study was that a novel ligament-derived matrix (LDM) would enhance expression of a ligamentous phenotype in hASCs compared to collagen gel alone. LDM prepared using phosphate-buffered saline or 0.1% peracetic acid was mixed with collagen gel (COL) and was evaluated for its ability to induce proliferation, differentiation, and extracellular matrix synthesis in hASCs over 28 days in culture at different seeding densities (0, 0.25 x 10(6), 1 x 10(6), or 2 x 10(6) hASC/mL). Biochemical and gene expression data were analyzed using analysis of variance. Fisher's least significant difference test was used to determine differences between treatments following analysis of variance. hASCs in either LDM or COL demonstrated changes in gene expression consistent with ligament development. hASCs cultured with LDM demonstrated more dsDNA content, sulfated-glycosaminoglycan accumulation, and type I and III collagen synthesis, and released more sulfated-glycosaminoglycan and collagen into the medium compared to hASCs in COL (p <or= 0.05). Increased seeding density increased DNA content incrementally over 28 days in culture for LDM but not COL constructs (p <or= 0.05). These findings suggest that LDM can stimulate a ligament phenotype by hASCs, and may provide a novel scaffold material for ligament engineering applications.
| S-EPMC2947935 | biostudies-literature
Project description:The tissues or organs derived decellularized extracellular matrix carry immunogenicity and the risk of pathogen transmission, resulting in limited therapeutic effects. The cell derived dECM cultured in vitro can address these potential risks, but its impact on wound remodeling is still unclear. This study aimed to explore the role of decellularized extracellular matrix (dECM) extracted from adipose derived stem cells (ADSCs) in skin regeneration. Methods: ADSCs were extracted from human adipose tissue. Then we cultivated adipose-derived stem cell cells and decellularized ADSC-dECM for freeze-drying. Western blot (WB), enzyme-linked immunosorbent assay (ELISA) and mass spectrometry (MS) were conducted to analyzed the main protein components in ADSC-dECM. The cell counting assay (CCK-8) and scratch assay were used to explore the effects of different concentrations of ADSC-dECM on the proliferation and migration of human keratinocytes cells (HaCaT), human umbilical vein endothelia cells (HUVEC) and human fibroblasts (HFB), respectively. Moreover, we designed a novel ADSC-dECM-CMC patch which used carboxymethylcellulose (CMC) to load with ADSC-dECM; and we further investigated its effect on a mouse full thickness skin wound model. Results: ADSC-dECM was obtained after decellularization of in vitro cultured human ADSCs. Western blot, ELISA and mass spectrometry results showed that ADSC-dECM contained various bioactive molecules, including collagen, elastin, laminin, and various growth factors. CCK-8 and scratch assay showed that ADSC-dECM treatment could significantly promote the proliferation and migration of HaCaT, human umbilical vein endothelia cells, and human fibroblasts, respectively. To evaluate the therapeutic effect on wound healing in vivo, we developed a novel ADSC-dECM-CMC patch and transplanted it into a mouse full-thickness skin wound model. And we found that ADSC-dECM-CMC patch treatment significantly accelerated the wound closure with time. Further histology and immunohistochemistry indicated that ADSC-dECM-CMC patch could promote tissue regeneration, as confirmed via enhanced angiogenesis and high cell proliferative activity. Conclusion: In this study, we developed a novel ADSC-dECM-CMC patch containing multiple bioactive molecules and exhibiting good biocompatibility for skin reconstruction and regeneration. This patch provides a new approach for the use of adipose stem cells in skin tissue engineering.
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