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Background: The beneficial effect of thermogenic adipocytes in maintaining body weight and protecting against metabolic disorders has raised interest in understanding the regulatory mechanisms defining white and beige adipocyte identity. Although alternative splicing has been shown to propagate adipose browning signals in mice, this has yet to be thoroughly investigated in human adipocytes. Methods: We performed parallel white and beige adipogenic differentiation using primary adipose stem cells from 6 unrelated healthy subjects and assessed differential gene and isoform expression in mature adipocytes by RNA sequencing. Results: We find 777 exon junctions with robust differential usage between white and beige adipocytes in all 6 subjects, mapping to 562 genes. Importantly, only 10% of these differentially spliced genes are also differentially expressed, indicating that alternative splicing constitutes an additional layer of gene expression regulation during beige adipocyte differentiation. Functional classification of alternative isoforms points to a gain of function for key thermogenic transcription factors such as PPARG and CITED1, and enzymes such as PEMT, or LPIN1. We find that a large majority of the splice variants arise from differential TSS usage, with beige-specific TSSs being enriched for PPARγ and MED1 binding compared to white-specific TSSs. Finally, we validate beige specific isoform expression at the protein level for two thermogenic regulators, PPARγ and PEMT. Discussion: These results suggest that differential isoform expression through alternative TSS usage is an important regulatory mechanism for human adipocyte thermogenic specification.
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Adipócitos Bege , Processamento Alternativo , Isoformas de Proteínas , Termogênese , Humanos , Adipócitos Bege/metabolismo , Termogênese/genética , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Diferenciação Celular , Adipogenia/genética , Masculino , Feminino , Adulto , Células Cultivadas , Regulação da Expressão Gênica , PPAR gama/genética , PPAR gama/metabolismoRESUMO
Alcohol dehydrogenase 1B (ADH1B) is a primate-specific enzyme which, uniquely among the ADH class 1 family, is highly expressed both in adipose tissue and liver. Its expression in adipose tissue is reduced in obesity and increased by insulin stimulation. Interference with ADH1B expression has also been reported to impair adipocyte function. To better understand the role of ADH1B in adipocytes, we used CRISPR/Cas9 to delete ADH1B in human adipose stem cells (ASC). Cells lacking ADH1B failed to differentiate into mature adipocytes manifested by minimal triglyceride accumulation and a marked reduction in expression of established adipocyte markers. As ADH1B is capable of converting retinol to retinoic acid (RA), we conducted rescue experiments. Incubation of ADH1B-deficient preadipocytes with 9-cis-RA, but not with all-transretinol, significantly rescued their ability to accumulate lipids and express markers of adipocyte differentiation. A homozygous missense variant in ADH1B (p.Arg313Cys) was found in a patient with congenital lipodystrophy of unknown cause. This variant significantly impaired the protein's dimerization, enzymatic activity, and its ability to rescue differentiation in ADH1B-deficient ASC. The allele frequency of this variant in the Middle Eastern population suggests that it is unlikely to be a fully penetrant cause of severe lipodystrophy. In conclusion, ADH1B appears to play an unexpected, crucial and cell-autonomous role in human adipocyte differentiation by serving as a necessary source of endogenous retinoic acid.
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Adipócitos , Adipogenia , Álcool Desidrogenase , Humanos , Álcool Desidrogenase/metabolismo , Álcool Desidrogenase/genética , Adipogenia/genética , Adipócitos/metabolismo , Adipócitos/citologia , Tretinoína/metabolismo , Diferenciação Celular , Sistemas CRISPR-Cas , Mutação de Sentido Incorreto , Tecido Adiposo/metabolismoRESUMO
Tendon tears are common and healing often occurs incompletely and by fibrosis. Tissue engineering seeks to improve repair, and one approach under investigation uses cell-seeded scaffolds containing biomimetic factors. Retention of biomimetic factors on the scaffolds is likely critical to maximize their benefit, while minimizing the risk of adverse effects, and without losing the beneficial effects of the biomimetic factors. The aim of the current study was to evaluate cross-linking methods to enhance the retention of tendon-derived matrix (TDM) on electrospun poly(ε-caprolactone) (PCL) scaffolds. We tested the effects of ultraviolet (UV) or carbodiimide (EDC:NHS:COOH) crosslinking methods to better retain TDM to the scaffolds and stimulate tendon-like matrix synthesis. Initially, we tested various crosslinking configurations of carbodiimide (2.5:1:1, 5:2:1, and 10:4:1 EDC:NHS:COOH ratios) and UV (30 s 1 J/cm2 , 60 s 1 J/cm2 , and 60 s 4 J/cm2 ) on PCL films compared to un-crosslinked TDM. We found that no crosslinking tested retained more TDM than coating alone (Kruskal-Wallis: p > .05), but that human adipose stem cells (hASCs) spread most on the 60 s 1 J/cm2 UV- and 2.5:1:1 EDC-crosslinked films (Kruskal-Wallis: p < .05). Next, we compared the effects of 60 s 1 J/cm2 UV- and 2.5:1:1 EDC-crosslinked to TDM-coated and untreated PCL scaffolds on hASC-induced tendon-like differentiation. UV-crosslinked scaffolds had greater modulus and stiffness than PCL or TDM scaffolds, and hASCs spread more on UV-crosslinked scaffolds (ANOVA: p < .05). Fourier transform infrared spectra revealed that UV- or EDC-crosslinking TDM did not affect the peaks at wavenumbers characteristic of tendon. Crosslinking TDM to electrospun scaffolds improves tendon-like matrix synthesis, providing a viable strategy for improving retention of TDM on electrospun PCL scaffolds.
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Colágeno , Engenharia Tecidual , Humanos , Engenharia Tecidual/métodos , Adipócitos , Tendões , Carbodi-Imidas , Alicerces Teciduais , PoliésteresRESUMO
Objective: Articular cartilage damages do not repair spontaneously. Tissue engineering is a promising approach to repair cartilage damage. Transforming growth factor-beta (TGF-ß) members are the known induction factors in chondrogenic differentiation. However, hypertrophy of the chondrocytes resulting from mesenchymal stem cells (MSCs) induction by TGF-ß is inevitable. Pomegranate fruit contains many ingredients which are useful in ensuring the health of organs. This study was designed to investigate the Pomegranate Fruit hydroalchoholic Extract (PFE) capability in human adipose derived stem cells (hASCs) differentiation into the chondrocytes on fibrin scaffold. Materials and Methods: Pomegranate fruit hydroalchoholic extract (PFE) was prepared. hASCs were isolated, expanded, labeled, and seeded on the fibrin scaffold. The constructs were divided into three groups including TGF-ß3, PFE, and control. The constructs were induced for 14 days, then, the MTT assay, Real-Time Polymerase Chain Reaction (PCR), and histochemistry assessments were run, and finally, the constructs were transplanted into the knee defect of rats. The gross and histological assessments of the transplants were done after 8 weeks. Results: The viability rate, COL2A1, Aggrecan (ACAN) and COL10A1 genes expression levels, and histological criterion of the PFE samples were significantly higher than that of the control. The macroscopic grades and histological results of the PFE samples were close to that of the TGF-ß3. The number of positive cells for COLÐI protein were higher significantly in the PFE group than the control. Conclusion: PFE was effective in the chondrogenic induction of hASCs. Further studies are needed to find out the events of the chondrogenic induction using PFE.
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Schwann cells (SCs) are myelinating cells that promote peripheral nerve regeneration. When nerve lesions form, SCs are destroyed, ultimately hindering nerve repair. The difficulty in treating nerve repair is exacerbated due to SC's limited and slow expansion capacity. Therapeutic use of adipose-derived stem cells (ASCs) is emerging in combating peripheral nerve injury due to these cells' SC differentiation capability and can be harvested easily in large numbers. Despite ASC's therapeutic potential, their transdifferentiation period typically takes more than two weeks. In this study, we demonstrate that metabolic glycoengineering (MGE) technology enhances ASC differentiation into SCs. Specifically, the sugar analog Ac5ManNTProp (TProp), which modulates cell surface sialylation, significantly improved ASC differentiation with upregulated SC protein S100ß and p75NGFR expression and elevated the neurotrophic factors nerve growth factor beta (NGFß) and glial cell-line-derived neurotrophic factor (GDNF). TProp treatment remarkably reduced the SC transdifferentiation period from about two weeks to two days in vitro, which has the potential to improve neuronal regeneration and facilitate future use of ASCs in regenerative medicine.
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Adipócitos , Células de Schwann , Humanos , Nervos Periféricos , Diferenciação Celular/fisiologia , Células-TroncoRESUMO
3D printing of polymeric scaffolds and autologous stem cells is a promising tool for damaged facial cartilage reconstruction surgeries. To this end, suitable bioinks are needed to generate scaffolds with the required morphological and functional features. We formulated hydrogel bioinks using k-Carrageen (kC) and poly(vinyl alcohol) (PVA) in three different weight ratios. The kC gives the systems the ability to undergo rapid sol-to-gel transitions upon cooling from 60 °C and above to body temperature, while the PVA is used as rheology modifier and porogen. The latter is crosslinked after molding or printing by freeze-thaw cycling for 1 day (FT1) or 5 days (FT5). To select the most suitable formulation for 3D printing, the sol-to-gel transition and the physico-chemical, mechanical and morphological properties of obtained hydrogels were studied. Moreover, the absence of cytotoxic effects of the material on SASCs was assessed in both stemness-preserving or chondro-inductive media. Printing trials were performed to identify optimal process parameters and co-printing and post-printing seeding approaches of SASCs were evaluated. Cells were found to be viable after co-printing and also after the FT1 treatment. Viable adherent cells were also found in the FT5 system, where cells were plated after freezing and thawing treatment.
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Impressão Tridimensional , Alicerces Teciduais , Carragenina/farmacologia , Carragenina/química , Alicerces Teciduais/química , Hidrogéis/farmacologia , Hidrogéis/química , Cartilagem , Engenharia TecidualRESUMO
The capacity of adipose stem/progenitor cells (ASCs) to undergo self-renewal and differentiation is crucial for adipose tissue homoeostasis, regeneration and expansion. However, the heterogeneous ASC populations of the adipose lineage constituting adipose tissue are not precisely known. In the present study, we demonstrate that cell surface expression of dipeptidyl peptidase-4 (DPP4)/cluster of differentiation 26 (CD26) subdivides the DLK1-/CD34+/CD45-/CD31- ASC pool of human white adipose tissues (WATs) into two large populations. Ex vivo, DPP4+ ASCs possess higher self-renewal and proliferation capacity and lesser adipocyte differentiation potential than DDP4- ASCs. The knock-down of DPP4 in ASC leads to significantly reduced proliferation and self-renewal capacity, while adipogenic differentiation is increased. Ectopic overexpression of DPP4 strongly inhibits adipogenesis. Moreover, in whole mount stainings of human subcutaneous (s)WAT, we detect DPP4 in CD34+ ASC located in the vascular stroma surrounding small blood vessels and in mature adipocytes. We conclude that DPP4 is a functional marker for an abundant ASC population in human WAT with high proliferation and self-renewal potential and low adipogenic differentiation capacity.
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Adipócitos , Dipeptidil Peptidase 4 , Adipócitos/metabolismo , Adipogenia , Tecido Adiposo/metabolismo , Tecido Adiposo Branco/metabolismo , Antígenos CD34/metabolismo , Diferenciação Celular , Dipeptidil Peptidase 4/genética , Dipeptidil Peptidase 4/metabolismo , Humanos , Células-Tronco/metabolismoRESUMO
Chronic recalcitrant wounds result from delayed or slowed healing processes. Underlying inflammation is a substantial risk factor for impaired dermal wound healing and often leads to chronic wound-related sequelae. Human adipose stem cells (hASCs) have shown tremendous potential in regenerative medicine. The goal of this project was to improve the outcome of chronic wounds by harvesting the exosomes from hASCs for therapeutic intervention. The results demonstrate that long noncoding RNA GAS5 is highly enriched in hASC exosomes and, further, that GAS5 is central to promoting wound repair in vitro. To evaluate the outcome of wound healing in a chronic low-grade inflammatory environment, lipopolysaccharide-treated HDF cells were evaluated for their response to hASC exosome treatment. Ingenuity pathway analysis identified inflammation pathways and genes affected by exosomes in a GAS5-dependent manner. Using siRNA to deplete GAS5 in HDF, the results demonstrated that Toll-like receptor 7 (TLR7) expression levels were regulated by GAS5. Importantly, the results demonstrate that GAS5 regulates inflammatory pathway genes in a chronic inflammation environment. The results presented here demonstrate that hASC exosomes are a viable therapeutic that accelerate the healing of chronic recalcitrant wounds.
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INTRODUCTION: Cardiovascular disease is the second killer across the globe, while coronary disease is the major cause. Cell therapy is one alternative to regenerate the infarcted heart wall. MATERIALS AND METHODS: In this study, the cardiomyogenesis capacity of human adipose stem cells (hAdSC) and human cardiomyocytes (hCardio) cultured in a 3-D biological scaffold (decellularised amnion bilayer) for nine days in a static condition was investigated. The cardiomyogenesis capacity of hAdSC were identified using immunohistochemistry and RT-PCR. The population of the cells isolated from the heart tissue expressed cTnT-1 (13.38 ± 11.38%), cKit (7.85 ± 4.2%), ICAM (85.53 ± 8.69%), PECAM (61.63 ± 7.18%) and VCAM (35.9 ± 9.11%), while from the fat tissue expressed the mesenchymal phenotypes (CD73, CD90, CD105, but not CD45, CD34, CD11b, CD19 and HLA-DR). Two age groups of hAdSC donors were compared, the youngsters (30-40yo) and the elderly (60-70 yo). RESULTS: The co-culture showed that after 5-day incubation, the seeded graft in the hAdSC-30 group had a tube-like appearance while the hAdSC-60 group demonstrated a disorganised pattern, despite of the MSC expressions of the hAdSC-60 were significantly higher. Initial co-culture showed no difference of ATP counts among all groups, however the hAdSC-30 group had the highest ATP count after 9 days culture (p = 0.004). After normalising to the normal myocardium, only the hAdSC-60 group expressed cTnT and MHC, very low, seen during the initial cultivation, but then disappeared. Meanwhile, the hAdSC-30 group expressed α-actinin, MHC and cTnT in the Day-5. The PPAR also was higher in the Day-5 compared to the Day-9 (p < 0.005). CONCLUSION: Cardiomyogenesis capacity of hAdSC co-cultured with hCardio in a 3-D scaffold taken from the 30-40yo donor showed better morphology and viability than the 60-70yo group, but maintained less than 5 days in this system.
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Âmnio , Células-Tronco Mesenquimais , Trifosfato de Adenosina/metabolismo , Células Cultivadas , Técnicas de Cocultura , Células-Tronco Mesenquimais/metabolismo , Miócitos CardíacosRESUMO
Severe injuries to skin including hypodermis require full-thickness skin replacement. Here, we bioengineered a tri-layered human skin substitute (TLSS) containing the epidermis, dermis, and hypodermis. The hypodermal layer was generated by differentiation of human adipose stem cells (ASC) in a collagen type I hydrogel and combined with a prevascularized dermis consisting of human dermal microvascular endothelial cells and fibroblasts, which arranged into a dense vascular network. Subsequently, keratinocytes were seeded on top to generate the epidermal layer of the TLSS. The differentiation of ASC into adipocytes was confirmed in vitro on the mRNA level by the presence of adiponectin, as well as by the expression of perilipin and FABP-4 proteins. Moreover, functional characteristics of the hypodermis in vitro and in vivo were evaluated by Oil Red O, BODIPY, and AdipoRed stainings visualizing intracellular lipid droplets. Further, we demonstrated that both undifferentiated ASC and mature adipocytes present in the hypodermis influenced the keratinocyte maturation and homeostasis in the skin substitutes after transplantation. In particular, an enhanced secretion of TGF-ß1 by these cells affected the epidermal morphogenesis as assessed by the expression of key proteins involved in the epidermal differentiation including cytokeratin 1, 10, 19 and cornified envelope formation such as involucrin. Here, we propose a novel functional hypodermal-dermo-epidermal tri-layered skin substitute containing blood capillaries that efficiently promote regeneration of skin defects. STATEMENT OF SIGNIFICANCE: The main objective of this study was to develop and assess the usefulness of a tri-layered human prevascularized skin substitute (TLSS) containing an epidermis, dermis, and hypodermis. The bioengineered hypodermis was generated from human adipose mesenchymal stem cells (ASC) and combined with a prevascularized dermis and epidermis. The TLSS represents an exceptional model for studying the role of cell-cell and cell-matrix interactions in vitro and in vivo. In particular, we observed that enhanced secretion of TGF-ß1 in the hypodermis exerted a profound impact on fibroblast and keratinocyte differentiation, as well as epidermal barrier formation and homeostasis. Therefore, improved understanding of the cell-cell interactions in such a physiological skin model is essential to gain insights into different aspects of wound healing.
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Pele Artificial , Bioengenharia , Derme , Células Endoteliais , Fibroblastos , Humanos , Queratinócitos , Pele , Tela Subcutânea , Engenharia TecidualRESUMO
Synergistic promotion of angiogenesis and osteogenesis in bone tissue-engineered constructs remains a crucial clinical challenge, which might be overcome by simultaneous employment of superior techniques including coculture systems, differentiation-stimulated factors, combinatorial scaffolds and bioreactors.Current study investigated the effect of flow perfusion along with coculture of human adipose stem cells (hASCs) and human umbilical vein endothelial cells (HUVECs) on osteogenic and angiogenic differentiation.Pre-treated hASCs with 1,25-dihydroxyvitamin D3 were seeded onto poly(lactic-co-glycolic acid)/ß-tricalcium phosphate/polycaprolactone (PLGA/ß-TCP/PCL) scaffold with/without HUVECs, and cultured for 14 days within a flask or modified perfusion bioreactor. Analysis of osteogenic and angiogenic gene expression, alkaline phosphatase (ALP) activity and ALP staining indicates a synergistic effect of perfusion flow and coculture system on osteogenic and angiogenic differentiation. The advantage of modified perfusion bioreactor is its five-branch flow distributor which directly connect to the porous PCL hollow fibers embedded in the 3D scaffold to improve flow and flow-induced shear stress uniformity.Dynamic coculture increased VEGF165 by 6-fold, VEGF189 by 2-fold, and Endothelin-1 by 4-fold, relative to dynamic monoculture. Static coculture enhanced osteogenic and angiogenic differentiation, compared with static monoculture. Although dynamic coculture is in preference to static coculture due to significant increase in ALP activity and promoted angiogenic marker expression. Our finding is the first to indicate that the modified perfusion bioreactor combined with the beneficial cell-cell crosstalk in pre-treated hASC/HUVEC cocultures provides a synergy between osteogenic and angiogenic differentiation of the accumulation of cells, suggesting that it represents a promising approach for regeneration of critical-sized bone defects.
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Osteogênese , Células-Tronco , Reatores Biológicos , Diferenciação Celular , Células Cultivadas , Técnicas de Cocultura , Células Endoteliais da Veia Umbilical Humana , Humanos , Perfusão , Alicerces TeciduaisRESUMO
Mesenchymal stem cells (MSCs) are multipotent stem cells with significant potential for regenerative medicine. The tumorigenesis of osteosarcoma is an intricate system and MSCs act as an indispensable part of this, interacting with the tumor microenvironment (TME) during the process. MSCs link to cells by acting on each component in the TME via autocrine or paracrine extracellular vesicles for cellular communication. Because of their unique characteristics, MSCs can be modified and processed into good biological carriers, loaded with drugs, and transfected with anticancer genes for the targeted treatment of osteosarcoma. Previous high-quality reviews have described the biological characteristics of MSCs; this review will discuss the effects of MSCs on the components of the TME and cellular communication and the prospects for clinical applications of MSCs.
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Human adipose stem cells (hASCs) are promising candidates for cell-based therapies, but they need to be efficiently expanded in vitro as they cannot be harvested in sufficient quantities. Recently, dynamic bioreactor systems operated with microcarriers achieved considerable high cell densities. Thus, they are a viable alternative to static planar cultivation systems to obtain high numbers of clinical-grade hASCs. Nevertheless, the production of considerable biomass in a short time must not be achieved to the detriment of the cells' quality. To facilitate the scalable expansion of hASC, we have developed a new serum- and xeno-free medium (UrSuppe) and a biodegradable microcarrier (BR44). In this study, we investigated whether the culture of hASCs in defined serum-free conditions on microcarriers (3D) or on planar (2D) cell culture vessels may influence the expression of some marker genes linked with the immature degree or the differentiated status of the cells. Furthermore, we investigated whether the biomaterials, which form our biodegradable MCs, may affect cell behavior and differentiation. The results confirmed that the quality and the undifferentiated status of the hASCs are very well preserved when they grow on BR44 MCs in defined serum-free conditions. Indeed, the ASCs showed a gene expression profile more compatible with an undifferentiated status than the same cells grown under standard planar conditions.
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Human adipose tissue-derived mesenchymal stem cells (hADSCs) due to easy extraction, relative abundance, in vitro expansion and differentiation potential, frozen storage capability, and ability to secrete cytokines, compared to other stem cells, are appropriate candidate in regenerative medicine. Extremely low-frequency electromagnetic fields (ELF-EMF) and betaine are two safe factors in bone lesions repair. This study was designed to assess the osteogenic differentiation potential of these factors on hADSCs. The samples were collected from women undergoing liposuction after obtaining written consent. The hADSCs were extracted and treated with osteogenesis differentiation medium (OD) as the positive control, with OD and betaine (BET group), with OD and EMF (EMF group), and with OD and betaine and EMF (BET+EMF group) for 21 d; the negative control consisted of cells without treatment. Betaine 10 mM and EMF with 50-Hz frequency, 1-mT intensity (8 h daily), and in the form of sinus wave were used. Osteogenic differentiation was evaluated by Alizarin Red staining, alkaline phosphatase activity, calcium deposition, and real-time PCR. A significant increase in calcium deposition in the BET+EMF group was observed compared to the other groups. The activity of alkaline phosphatase in the positive control and BET groups was increased significantly compared to EMF and BET + EMF groups and a significant increase of this enzyme activity in the BET + EMF compared to EMF group was observed. The expression of RUNX2 and OCN genes in the EMF-treated groups were significantly reduced compared to the non-EMF-treated groups, and BET+EMF showed a significant increase of RUNX2 gene expression as compared the EMF group. The ELF-EMF leads to a decrease in the osteogenic differentiation and the expression RUNX2 and OCN genes in hADSCs. But osteogenic differentiation and RUNX2 gene expression were increased post-induction by betaine. The synergic effect of betaine and EMF on the osteogenic differentiation and related genes expression of hADSCs was higher than EMF.
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Betaína/farmacologia , Diferenciação Celular/genética , Campos Eletromagnéticos , Osteogênese/genética , Fosfatase Alcalina/genética , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/efeitos da radiação , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/efeitos da radiação , Células Cultivadas , Humanos , Células-Tronco Mesenquimais/efeitos dos fármacos , Células-Tronco Mesenquimais/efeitos da radiação , Osteogênese/efeitos dos fármacos , Osteogênese/efeitos da radiaçãoRESUMO
One of the promising approaches for the treatment of cardiac disease is stem cell therapy. In this study, we compared the cardiomyogenic differentiation rate, from human adipose-derived stem cells (hADSCs) in a three-dimensional (3D) hanging drop (HD) spheroid culture system, versus a two-dimensional (2D) culture condition at different concentrations of 5-azacytidine (5-Aza). 5-Azaytidine (5-Aza) is a pyrimidine nucleoside analogue of cytidine that initiates cell differentiation programs through DNA demethylation. The hADSCs were isolated and cultured both in 2D and 3D HD conditions, with either 10 or 50 µM concentrations of 5-Aza. Then DNA content, gene expression, and protein content were analyzed. 3D HD culture resulted in a higher percentage of cells in G0/G1 and S phase in the cell division cycle, whereas 2D culture led to a greater percentage of cells in the G2/M phase. A significantly higher gene expression rate of HAND1, HAND2, cTnI, Cx43, ßMHC, GATA4, NKX2.5, and MLC2V was observed in HD treated with 50 µM 5-Aza. This was confirmed by immunocytochemistry. These findings suggest that 50 µM concentration of 5-Aza can induce hADSCs to differentiate into cardiomyocytes. The differentiation rate was significantly higher when accompanied by the 3D HD culture system. This work provides a new culture system for cell differentiation for cardiovascular tissue engineering.
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Tecido Adiposo/metabolismo , Azacitidina/farmacologia , Diferenciação Celular/efeitos dos fármacos , Células-Tronco Mesenquimais/metabolismo , Miócitos Cardíacos/metabolismo , Tecido Adiposo/citologia , Técnicas de Cultura de Células , Células Cultivadas , Feminino , Humanos , Células-Tronco Mesenquimais/citologia , Pessoa de Meia-Idade , Miócitos Cardíacos/citologiaRESUMO
Adipose-derived stem cells (ASCs) reside in the stromal compartment of adipose tissue and can be easily harvested in large quantities through a clinically safe liposuction procedure. ASCs do not induce immunogenic reactions and rather exert immunosuppressive effects. Therefore, they can be used for both autologous and allogeneic transplantations. They hold great promise for cell-based therapies and tissue engineering. A prerequisite to the realization of this promise is the development of successful cryopreservation methods for ASCs. In this chapter, we describe a xeno-free- and chemically defined cryopreservation protocol, which can be used for various clinical applications of ASCs.
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Adipócitos/citologia , Técnicas de Cultura de Células/métodos , Diferenciação Celular , Criopreservação/métodos , Crioprotetores/farmacologia , Células-Tronco/citologia , Engenharia Tecidual/métodos , Adipócitos/efeitos dos fármacos , Proliferação de Células , Células Cultivadas , Humanos , Células-Tronco/efeitos dos fármacosRESUMO
Studies using polymeric scaffolds for various biomedical applications, such as tissue engineering, implants and medical substitutes, and drug delivery systems, have attempted to identify suitable material for tissue regeneration. This study aimed to investigate the biocompatibility and effectiveness of a gelatin scaffold seeded with human adipose stem cells (hASCs), including physical characteristics, multilineage differentiation in vitro, and osteogenic potential, in a rat model of a calvarial bone defect and to optimize its design. This functionalized scaffold comprised gelatin-hASCs layers to improve their efficacy in various biomedical applications. The gelatin scaffold exhibited excellent biocompatibility in vitro after two weeks of implantation. Furthermore, the gelatin scaffold supported and specifically regulated the proliferation and osteogenic and chondrogenic differentiation of hASCs, respectively. After 12 weeks of implantation, upon treatment with the gelatin-hASCs scaffold, the calvarial bone harboring the critical defect regenerated better and displayed greater osteogenic potential without any damage to the surrounding tissues compared to the untreated bone defect. These findings suggest that the present gelatin scaffold is a good potential carrier for stem cells in various tissue engineering applications.
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Diferenciação Celular , Células Imobilizadas , Gelatina/química , Transplante de Células-Tronco Mesenquimais , Células-Tronco Mesenquimais/metabolismo , Crânio , Engenharia Tecidual , Alicerces Teciduais/química , Animais , Células Imobilizadas/metabolismo , Células Imobilizadas/transplante , Xenoenxertos , Humanos , Masculino , Ratos , Ratos Sprague-Dawley , Crânio/lesões , Crânio/metabolismo , Crânio/patologiaRESUMO
Functional recovery after neurotmesis, a complete transection of the nerve fiber, is often poor and requires a surgical procedure. Especially for longer gaps (>3 mm), end-to-end suturing of the proximal to the distal part is not possible, thus requiring nerve graft implantation. Artificial nerve grafts, i.e., hollow fibers, hydrogels, chitosan, collagen conduits, and decellularized scaffolds hold promise provided that these structures are populated with Schwann cells (SC) that are widely accepted to promote peripheral and spinal cord regeneration. However, these cells must be collected from the healthy peripheral nerves, resulting in significant time delay for treatment and undesired morbidities for the donors. Therefore, there is a clear need to explore the viable source of cells with a regenerative potential similar to SC. For this, we analyzed the literature for the generation of Schwann cell-like cells (SCLC) from stem cells of different origins (i.e., mesenchymal stem cells, pluripotent stem cells, and genetically programmed somatic cells) and compared their biological performance to promote axonal regeneration. Thus, the present review accounts for current developments in the field of SCLC differentiation, their applications in peripheral and central nervous system injury, and provides insights for future strategies.
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Sistema Nervoso Central/fisiopatologia , Sistema Nervoso Periférico/fisiopatologia , Células de Schwann/metabolismo , Humanos , Regeneração Nervosa/fisiologiaRESUMO
Long-term primary culture of mammalian cells has been always difficult due to unavoidable senescence. Conventional methods for generating immortalized cell lines usually require manipulation of genome which leads to change of important biological and genetic characteristics. Recently, conditional reprogramming (CR) emerges as a novel next generation tool for long-term culture of primary epithelium cells derived from almost all origins without alteration of genetic background of primary cells. CR co-cultures primary cells with inactivated mouse 3T3-J2 fibroblasts in the presence of RHO-related protein kinase (ROCK) inhibitor Y-27632, enabling primary cells to acquire stem-like characteristics while retain their ability to fully differentiate. With only a few years' development, CR shows broad prospects in applications in varied areas including disease modeling, regenerative medicine, drug evaluation, drug discovery as well as precision medicine. This review is thus to comprehensively summarize and assess current progress in understanding mechanism of CR and its wide applications, highlighting the value of CR in both basic and translational researches and discussing the challenges faced with CR.
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We report on a potential strategy involving the exogenous neurotrophic factors (NTF) for enhancing the neurotrophic capacity of human adipose stem cells (ASC) in vitro. For this, ASC were stimulated for three days using NTF, i.e., nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin 3 (NT3), NT4, glial cell-derived neurotrophic factor (GDNF), and ciliary neurotrophic factor (CNTF). The resulting conditioned medium (CM) as well as individual NTF exhibited distinct effects on axonal outgrowth from dorsal root ganglion (DRG) explants. In particular, CM derived from NT3-stimulated ASC (CM-NT3-ASC) promoted robust axonal outgrowth. Subsequent transcriptional analysis of DRG cultures in response to CM-NT3-ASC displayed significant upregulation of STAT-3 and GAP-43. In addition, phosphoproteomic analysis of NT3-stimulated ASC revealed significant changes in the phosphorylation state of different proteins that are involved in cytokine release, growth factors signaling, stem cell maintenance, and differentiation. Furthermore, DRG cultures treated with CM-NT3-ASC exhibited significant changes in the phosphorylation levels of proteins involved in tubulin and actin cytoskeletal pathways, which are crucial for axonal growth and elongation. Thus, the results obtained at the transcriptional, proteomic, and cellular level reveal significant changes in the neurotrophic capacity of ASC following NT3 stimulation and provide new options for improving the axonal growth-promoting potential of ASC in vitro.