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Enhanced cellular engraftment of adipose-derived mesenchymal stem cell spheroids by using nanosheets as scaffolds.

ABSTRACT: The short survival time of transplanted adipose-derived mesenchymal stem cells (ASCs) is a problem for skin wound healing. Transplantation after the formation of cellular spheroids has been investigated as a promising method for prolonging cellular survival. However, there have been technical restrictions for transplantation of spheroids in clinical practice. Here, we show an effective method for transplantation of ASC spheroids onto skin wounds in order to efficiently cure refractory ulcers. To assist anchoring of spheroids onto skin wounds, we used a 120-nm-thick free-standing film (nanosheet) that has a highly adhesive property. Bioluminescence imaging showed that ASC spheroids carried by the nanosheet survived for 14 days, which is about two-times longer than that previously reported. Wounds treated with a nanosheet carrying ASC spheroids were 4-times smaller than untreated wounds on day 14. This method for transplantation of spheroids could be applied to cell therapy for various refractory skin wounds.

SUBMITTER: Nagano H

PROVIDER: S-EPMC8280158 | biostudies-literature | 2021 Jul

REPOSITORIES: biostudies-literature

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Enhanced cellular engraftment of adipose-derived mesenchymal stem cell spheroids by using nanosheets as scaffolds.

Nagano Hisato H Suematsu Yoshitaka Y Takuma Megumi M Aoki Shimpo S Satoh Ayano A Takayama Eiji E Kinoshita Manabu M Morimoto Yuji Y Takeoka Shinji S Fujie Toshinori T Kiyosawa Tomoharu T

Scientific reports 20210714 1

The short survival time of transplanted adipose-derived mesenchymal stem cells (ASCs) is a problem for skin wound healing. Transplantation after the formation of cellular spheroids has been investigated as a promising method for prolonging cellular survival. However, there have been technical restrictions for transplantation of spheroids in clinical practice. Here, we show an effective method for transplantation of ASC spheroids onto skin wounds in order to efficiently cure refractory ulcers. To ...[more]

PMID: 34262089

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Project description:IntroductionEngineered bone graft substitutes are a promising alternative and supplement to autologous bone grafts as treatments for bone healing impairment. Advances in human medicine extend an invitation to pursue these biomimetic strategies in animal patients, substantiated by the theory that specialized scaffolds, multipotent cells, and biological cues may be combined into a bioactive implant intended for the enhancement of tissue regeneration.MethodsThis proof-of-concept study was designed to evaluate and validate the feasibility of beta-tricalcium phosphate foam scaffolds seeded with canine mesenchymal stem cells derived from adipose tissue. Cell-inoculated samples and sham controls were cultured statically for 72 hours in complete growth medium to evaluate seeding capacity, while a subset of loaded scaffolds was further induced with osteogenic culture medium for 21 days. Produced implants were characterized and validated with a combination of immunofluorescence and reflection confocal microscopy, scanning electron microscopy, and polymerase chain reaction to confirm osteogenic differentiation in tridimensional-induced samples.ResultsAfter 72 hours of culture, all inoculated scaffolds presented widespread yet heterogeneous surface seeding, distinctively congregating stem cells around pore openings. Furthermore, at 21 days of osteogenic culture conditions, robust osteoblastic differentiation of the seeded cells was confirmed by the change of cell morphology and evident deposition of extra-cellular matrix, accompanied by mineralization and scaffold remodeling; furthermore, all induced cell-loaded implants lost specific stemness immunophenotype expression and simultaneously upregulated genomic expression of osteogenic genes Osterix and Ostecalcin.Conclusionsβ-TCP bio-ceramic foam scaffolds proved to be suitable carriers and hosts of canine adipose-derived MSCs, promoting not only surface attachment and proliferation, but also demonstrating strong in-vitro osteogenic potential. Although this research provides satisfactory in-vitro validation for the conceptualization and feasibility of a canine bio-active bone implant, further testing such as patient safety, large-scale reproducibility, and quality assessment are needed for regulatory compliance in future commercial clinical applications.

Project description:IntroductionDifferentiation of hepatocytes and culture methods have been investigated in dogs as a tool to establish liver transplant and drug metabolism examination systems. However, mass culture techniques for canine hepatocytes (cHep) have not been investigated, and it is necessary to construct a suitable culture system. Recently, a protocol called Bud production has attracted attention, and a mixed culture of human and mouse hepatocytes, stem cells, and artificial blood vessels significantly improved the size and formation ratio of spheroids. The purpose of this study was to investigate and improve the in vitro culture of cHep by mixing canine adipose-derived mesenchymal stem cells (cASCs) and human umbilical vein endothelial cells (HUVECs).MethodsSpheroid formation ratio and histological examination were evaluated among four culture methods, including cHep alone, two-mix (cHep + cASCs and cHep + HUVEC), and three-mix (cHep + HUVEC + cASCs), on days 0, 4, and 7. Expression levels of liver-related genes (ALB, AFP, α1-AT, CDH1, CYP2E1, CYP3A12, and TAT) were evaluated by quantitative real-time polymerase chain reaction (RT-PCR). Protein expression of albumin, vimentin, and von Willebrand Factor (vWF) was investigated to confirm the location of the hepatocytes.ResultsThe ratio of spheroid formation was 60.2% in the three-mix culture and was significantly improved compared with cHep alone (5.9%) and two-mix; cHep + cASCs (36.2%) and cHep + HUVEC (26.4%) (P < 0.001). Histological evaluation revealed that the three-mix spheroids formed large canine hepatocyte spheroids (LcHS), and hepatocytes were distributed in the center of the spheroids. Quantitative gene expression analysis of LcHS showed that liver-related genes expression were maintained the same levels with that of a culture of cHep alone from days 4-7.ConclusionThese results revealed that the three-mix culture method using cHep, HUVECs, and cASCs was capable of promoting LcHS without impairing liver function in cHep, suggesting that LcHS could be used for the application of high-volume culture techniques in dogs.

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Project description:BackgroundAdipose-derived stem cell (ASC) has been considered as a desirable source for cell therapy. In contrast to combining scaffold materials with cells, ASCs can be fabricated into scaffold-free three-dimensional (3D) constructs to promote regeneration at tissue level. However, previous reports have found decreased expression of vascular endothelial growth factor (VEGF) in ASC sheets. In this study, we aimed to integrate ASC spheroids into ASC sheets to enhance the angiogenic capability of cell sheets.MethodsASCs were seeded in agarose microwells to generate uniform cell spheroids with adjustable size, while extracellular matrix deposition could be stimulated by ascorbic acid 2-phosphate to form ASC sheets. RNA sequencing was performed to identify the transcriptomic profiles of ASC spheroids and sheets relative to monolayer ASCs. By transferring ASC spheroids onto ASC sheets, the spheroid sheet composites could be successfully fabricated after a short-term co-culture, and their angiogenic potential was evaluated in vitro and in ovo.ResultsRNA sequencing analysis revealed that upregulation of angiogenesis-related genes was found only in ASC spheroids. The stimulating effect of spheroid formation on ASCs toward endothelial lineage was demonstrated by enhanced CD31 expression, which maintained after ASC spheroids were seeded on cell sheets. Relative to ASC sheets, enhanced expression of VEGF and hepatocyte growth factor was also noted in ASC spheroid sheets, and conditioned medium of ASC spheroid sheets significantly enhanced tube formation of endothelial cells in vitro. Moreover, chick embryo chorioallantoic membrane assay showed a significantly higher capillary density with more branch points after applying ASC spheroid sheets, and immunohistochemistry also revealed a significantly higher ratio of CD31-positive area.ConclusionIn the spheroid sheet construct, ASC spheroids can augment the pro-angiogenesis capability of ASC sheets without the use of exogenous biomaterial or genetic manipulation. The strategy of this composite system holds promise as an advance in 3D culture technique of ASCs for future application in angiogenesis and regeneration therapies.

Dataset Information

Enhanced cellular engraftment of adipose-derived mesenchymal stem cell spheroids by using nanosheets as scaffolds.

Publications

Enhanced cellular engraftment of adipose-derived mesenchymal stem cell spheroids by using nanosheets as scaffolds.

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