Journal: Biomedicines

Article Title: SDF-1α Gene-Activated Collagen Scaffold Restores Pro-Angiogenic Wound Healing Features in Human Diabetic Adipose-Derived Stem Cells

doi: 10.3390/biomedicines9020160

Figure Lengend Snippet: Gene-activated scaffold for wound healing applications and its impact on human adipose-derived stem cells (ADSCs). ( A ) A schematic of the application of a gene-activated scaffold for wound healing. Gene-activated scaffolds can be directly implanted into the wound where host cells infiltrating the gene-activated scaffold induce the reparative effects. Alternatively, the tissue engineering approach can be adopted where stem cells are grown on the gene-activated scaffold and the resulting construct is implanted into the wound site. ( B ) Relative expression of functional factors produced by human diabetic ADSCs on the gene-free scaffold compared to healthy ADSCs on the gene-free scaffold on day 7. An angiogenesis proteome profiler was used to screen the contents of the ADSCs secretome. Conditioned media (CM) were pooled in equal volumes from the three replicates to assay the proteome. Expression levels were quantified based on mean volume intensities determined using a ChemiDoc system. The diabetic ADSCs on the gene-free scaffold produced elevated levels of anti-angiogenic factors (PAI-1, TIMP-1, PEDF and TSP-1), inflammatory cytokine IL-8 and vascular disruptive factor Ang-2 relative to their healthy equivalent. ( C ) Representative blot images of the proteome analysis.

Article Snippet: Human ADSCs (healthy, female, age 33, Cat no. 10HU-001, Lot no. 200359; diabetic type 2, female, age 45, Cat no.10HU-007, Lot no. 200404) purchased from iXCells Biotechnologies, were expanded to passage 4 in ADSCs growth medium (Cat no. MD0003) supplied by the company.

Techniques: Derivative Assay, Construct, Expressing, Functional Assay, Produced

Journal: Biomedicines

Article Title: SDF-1α Gene-Activated Collagen Scaffold Restores Pro-Angiogenic Wound Healing Features in Human Diabetic Adipose-Derived Stem Cells

doi: 10.3390/biomedicines9020160

Figure Lengend Snippet: Impact of SDF-1α gene-activated scaffold on the activation of SDF-1α and its downstream signaling mediators in diabetic ADSCs. ( A ) SDF-1α gene-activated scaffold caused the overexpression of SDF-1α mRNA in both healthy and diabetic ADSCs. The overexpression of SDF-1α mRNA had a minimal effect on the expression of CXCR7 mRNA in diabetic ADSCs, while it increased significantly in healthy ADSCs. ( B ) Immunofluorescence images showing the abundancy of SDF-1α and CXCR7 in the ADSCs groups. ( C ) The diabetic ADSCs on the SDF-1α gene-activated scaffold expressed the highest level of SDF-1α and CXCR7 proteins, while healthy ADSCs displayed the weakest expression of CXCR7. *, **, *** and **** indicate statistical significance at p < 0.05, p < 0.01, p < 0.005 and p < 0.001, respectively. Data are presented as mean ± standard deviation ( n = 3). Scale bar 20 μm.

Article Snippet: Human ADSCs (healthy, female, age 33, Cat no. 10HU-001, Lot no. 200359; diabetic type 2, female, age 45, Cat no.10HU-007, Lot no. 200404) purchased from iXCells Biotechnologies, were expanded to passage 4 in ADSCs growth medium (Cat no. MD0003) supplied by the company.

Techniques: Activation Assay, Over Expression, Expressing, Immunofluorescence, Standard Deviation

Journal: Biomedicines

Article Title: SDF-1α Gene-Activated Collagen Scaffold Restores Pro-Angiogenic Wound Healing Features in Human Diabetic Adipose-Derived Stem Cells

doi: 10.3390/biomedicines9020160

Figure Lengend Snippet: Impact of SDF-1α gene-activated scaffold on the production of functional factors in healthy and diabetic ADSCs. ( A ) Transfection of the diabetic ADSCs within the SDF-1α gene-activated scaffold resulted in restoration of a healthy-like signaling of functional factors in the diabetic ADSCs. On the other hand, the SDF-1α gene-activated scaffold caused a moderate deviation in the signaling pattern of the functional factors in the healthy ADSCs relative to its unactivated equivalent. ( B ) Representative blot images of the proteome analysis.

Article Snippet: Human ADSCs (healthy, female, age 33, Cat no. 10HU-001, Lot no. 200359; diabetic type 2, female, age 45, Cat no.10HU-007, Lot no. 200404) purchased from iXCells Biotechnologies, were expanded to passage 4 in ADSCs growth medium (Cat no. MD0003) supplied by the company.

Techniques: Functional Assay, Transfection

Journal: Biomedicines

Article Title: SDF-1α Gene-Activated Collagen Scaffold Restores Pro-Angiogenic Wound Healing Features in Human Diabetic Adipose-Derived Stem Cells

doi: 10.3390/biomedicines9020160

Figure Lengend Snippet: Pro-angiogenic impact of secreted factors from the transfected healthy and diabetic ADSCs. ( A ) The diabetic ADSCs on the SDF-1α gene-activated scaffold induced the strongest pro-angiogenic response in human endothelial cells. ( B ) At 8 h post-exposure, CM from diabetic ADSCs significantly enhanced endothelial network branching ( p < 0.01) as well as tubules formation ( p < 0.05), compared to that induced by their healthy counterpart. EndoGro Media (EGM) + VEGF was used as reference medium to stimulate endothelial angiogenesis. Scale bar 100 μm. * p < 0.01, ** p < 0.05.

Article Snippet: Human ADSCs (healthy, female, age 33, Cat no. 10HU-001, Lot no. 200359; diabetic type 2, female, age 45, Cat no.10HU-007, Lot no. 200404) purchased from iXCells Biotechnologies, were expanded to passage 4 in ADSCs growth medium (Cat no. MD0003) supplied by the company.

Techniques: Transfection

Journal: Biomedicines

Article Title: SDF-1α Gene-Activated Collagen Scaffold Restores Pro-Angiogenic Wound Healing Features in Human Diabetic Adipose-Derived Stem Cells

doi: 10.3390/biomedicines9020160

Figure Lengend Snippet: Effect of SDF-1α gene-activated scaffold on the expression of pro-wound healing matrix genes in healthy and diabetic ADSCs. ( A ) On day 7, the transfected diabetic ADSCs on the gene-activated scaffold showed a significantly ( p < 0.05) enhanced transcription of the FN1 gene than the healthy ADSCs on the gene-free scaffold. ( B ) At the same time point, the healthy ADSCs on the gene-activated scaffold showed a significant activation of the COL4A1 gene than their gene-free equivalent. *, ** and *** indicate statistical significance at p < 0.05, p < 0.01 and p < 0.005, respectively. Data are presented as mean ± standard deviation ( n = 3).

Article Snippet: Human ADSCs (healthy, female, age 33, Cat no. 10HU-001, Lot no. 200359; diabetic type 2, female, age 45, Cat no.10HU-007, Lot no. 200404) purchased from iXCells Biotechnologies, were expanded to passage 4 in ADSCs growth medium (Cat no. MD0003) supplied by the company.

Techniques: Expressing, Transfection, Activation Assay, Standard Deviation

Journal: Biomedicines

Article Title: SDF-1α Gene-Activated Collagen Scaffold Restores Pro-Angiogenic Wound Healing Features in Human Diabetic Adipose-Derived Stem Cells

doi: 10.3390/biomedicines9020160

Figure Lengend Snippet: Effect of SDF-1α gene-activated scaffold on the deposition and remodeling of pro-wound healing matrix proteins in healthy and diabetic ADSCs. ( A ) Relative to the healthy ADSCs on the gene-free scaffold, diabetic ADSCs on the SDF-1α gene-activated scaffold showed a significant decrease in the deposition of fibronectin matrix while increasing the deposition of collagen IV over time. Contrarily, healthy ADSCs on the SDF-1α gene-activated scaffold deposited minimal amounts of the fibronectin matrix throughout the culture period but significantly increased the deposition of collagen IV. ( B ) Semi-quantitative interpretation of spatiotemporal expression of the matrix proteins. * and ** indicate statistical significance at p < 0.05 and p < 0.01, respectively. Data are presented as mean ± standard deviation ( n = 3). Scale bar 20 μm.

Article Snippet: Human ADSCs (healthy, female, age 33, Cat no. 10HU-001, Lot no. 200359; diabetic type 2, female, age 45, Cat no.10HU-007, Lot no. 200404) purchased from iXCells Biotechnologies, were expanded to passage 4 in ADSCs growth medium (Cat no. MD0003) supplied by the company.

Techniques: Expressing, Standard Deviation

Journal: Biomedicines

Article Title: SDF-1α Gene-Activated Collagen Scaffold Restores Pro-Angiogenic Wound Healing Features in Human Diabetic Adipose-Derived Stem Cells

doi: 10.3390/biomedicines9020160

Figure Lengend Snippet: A schematic of the functional changes induced by the SDF-1α gene-activated collagen scaffold in healthy and diabetic ADSCs. The diabetic ADSCs on the gene-free scaffold initially showed an impaired functional response characterized by elevated inflammatory cytokine production (IL-8), anti-angiogenic factors (PAI-1, TIMP-1, PEDF and TSP-1) and vascular destabilizing factor Ang-2 compared to the healthy ADSCs on the gene-free scaffold. However, when activated within the gene-activated scaffold, the impaired signaling in the diabetic ADSCs could be restored to a healthy-like state and exert therapeutic paracrine effects capable of enhancing angiogenesis. Meanwhile, the gene-activated scaffold drove the healthy ADSCs towards an advanced cellular maturation stage while facilitating the bypass of early signaling events such as the production of uPA, VEGF and MCP-1 and the deposition of the provisional matrix fibronectin.

Article Snippet: Human ADSCs (healthy, female, age 33, Cat no. 10HU-001, Lot no. 200359; diabetic type 2, female, age 45, Cat no.10HU-007, Lot no. 200404) purchased from iXCells Biotechnologies, were expanded to passage 4 in ADSCs growth medium (Cat no. MD0003) supplied by the company.

Techniques: Functional Assay

Journal: Bioengineered

Article Title: Small activating RNA activation of ATOH1 promotes regeneration of human inner ear hair cells

doi: 10.1080/21655979.2022.2045835

Figure Lengend Snippet: Screening of ATOH1 small activating RNAs in 293 T cells and mesenchymal stem cells . Cells were transfected with the indicated small activating RNAs using RNAiMAX at the indicated concentrations for 72 h. Mock samples were transfected in the absence of duplex RNA. Plasmid DNA was transfected at 1 µg/µl using Lipofectamine 3000. The transfected cells were subjected to RT–qPCR after RNA isolation, an RT reaction to generate cDNA and a Western blotting assay after protein isolation. A and B. Relative mRNA expression levels of the ATOH1 gene in 293 T cells (a) and mesenchymal stem cells (b) 72 h after transfection. C and D. ATOH1 protein levels assessed by Western blotting in 293 T cells (c) and mesenchymal stem cells (d) at 72 h after transfection. E and F. Relative ATOH1 mRNA expression levels in response to different small activating RNA concentrations.

Article Snippet: The human embryonic kidney cell line 293 T (ATCC® CRL) and human adipose-derived mesenchymal stem cells (ScienCell) were used as the cell lines to screen the small activating RNAs.

Techniques: Transfection, Plasmid Preparation, Quantitative RT-PCR, Isolation, Western Blot, Expressing

Journal: Bioengineered

Article Title: Small activating RNA activation of ATOH1 promotes regeneration of human inner ear hair cells

doi: 10.1080/21655979.2022.2045835

Figure Lengend Snippet: Induction of hair cell progenitor cells . Mesenchymal stem cells were cultured in induction medium containing EGF, IGF-1 and bFGF for 4 weeks, and hair cell progenitor cell marker genes were detected. A . Mesenchymal stem cells were cultured in hair cell progenitor cell induction medium as described in the Materials and Methods. Total cellular RNA was isolated from the treated cells and reverse transcribed into cDNA, which was amplified by semiquantitative RT–PCR. The GAPDH gene was also amplified as a control for RNA loading. B. Treated cells were fixed and stained with an antibody against PAX8, and the nuclei were counterstained. Red arrows denote positively stained cells.

Article Snippet: The human embryonic kidney cell line 293 T (ATCC® CRL) and human adipose-derived mesenchymal stem cells (ScienCell) were used as the cell lines to screen the small activating RNAs.

Techniques: Cell Culture, Marker, Isolation, Reverse Transcription, Amplification, Reverse Transcription Polymerase Chain Reaction, Control, Staining