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. 2022 Mar 28;20(1):164.
doi: 10.1186/s12951-022-01352-6.

Congenital microtia patients: the genetically engineered exosomes released from porous gelatin methacryloyl hydrogel for downstream small RNA profiling, functional modulation of microtia chondrocytes and tissue-engineered ear cartilage regeneration

Jianguo Chen  1 , Tianyu Huang  1 , Ruiquan Liu  1 , Chenyu Wang  1 , Haiyue Jiang  2 , Hengyun Sun  3
Affiliations

Congenital microtia patients: the genetically engineered exosomes released from porous gelatin methacryloyl hydrogel for downstream small RNA profiling, functional modulation of microtia chondrocytes and tissue-engineered ear cartilage regeneration

Jianguo Chen et al. J Nanobiotechnology. .

Abstract

Background: Mesenchymal stem cells (MSCs) exosomes were previously shown to be effective in articular cartilage repairing. However, whether MSCs exosomes promote mature cartilage formation of microtia chondrocytes and the underlying mechanism of action remains unknown. Additionally, some hurdles, such as the low yield and unsatisfactory therapeutic effects of natural exosomes have emerged when considering the translation of exosomes-therapeutics to clinical practices or industrial production. Herein, we investigated the roles of human adipose-derived stem cells (ADSCs) exosomes in modulating microtia chondrocytes and the underlying mechanism of action. Special attention was also paid to the mass production and functional modification of ADSCs exosomes.

Results: We firstly used porous gelatin methacryloyl (Porous Gelma) hydrogel with pores size of 100 to 200 μm for 3D culture of passage 2, 4 and 6 ADSCs (P2, P4 and P6 ADSCs, respectively), and obtained their corresponding exosomes (Exo 2, Exo 4 and Exo 6, respectively). In vitro results showed Exo 2 outperformed both Exo 4 and Exo 6 in enhancing cell proliferation and attenuating apoptosis. However, both Exo 4 and Exo 6 promoted chondrogenesis more than Exo 2 did. Small RNA sequencing results indicated Exo 4 was similar to Exo 6 in small RNA profiles and consistently upregulated PI3K/AKT/mTOR signaling pathway. Notably, we found hsa-miR-23a-3p was highly expressed in Exo 4 and Exo 6 compared to Exo 2, and they modulated microtia chondrocytes by transferring hsa-miR-23a-3p to suppress PTEN expression, and consequently to activate PI3K/AKT/mTOR signaling pathway. Then, we designed genetically engineered exosomes by directly transfecting agomir-23a-3p into parent P4 ADSCs and isolated hsa-miR-23a-3p-rich exosomes for optimizing favorable effects on cell viability and new cartilage formation. Subsequently, we applied the engineered exosomes to in vitro and in vivo tissue-engineered cartilage culture and consistently found that the engineered exosomes could enhance cell proliferation, attenuate apoptosis and promote cartilage regeneration.

Conclusions: Taken together, the porous Gelma hydrogel could be applied to exosomes mass production, and functional modification could be achieved by selecting P4 ADSCs as parent cells and genetically modifying ADSCs. Our engineered exosomes are a promising candidate for tissue-engineered ear cartilage regeneration.

Keywords: Adipose-derived stem cells; Engineered exosomes; Microtia; Porous gelatin methacryloyl; Tissue-engineered ear cartilage.

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Conflict of interest statement

The authors declare that they have no competing interests.

Figures

Fig. 1
Fig. 1
Identification of P2, P4 and P6 human ADSCs and characterization of porous Gelma hydrogel. A Representative images showing the spindle-like morphology of ADSCs in 2D culture dish. Scale bar: 50 μm. B Representative images showing the live and dead ADSCs within porous Gelma hydrogel, C and the rate of live cell. Scale bar: 500 μm. Green: live ADSCs. Red: dead ADSCs. D Characteristic surface markers of ADSCs evaluated by flow cytometry analysis. E Diagram showing 3D-Exo released from porous Gelma hydrogel. F Exosomes yield comparison between 2D- and 3D-cultured conditioned medium from the culture of P2, P4 and P6 ADSCs. G Profile of exosomes released from the porous Gelma hydrogel. H Swelling ratio of the porous Gelma hydrogel in PBS (pH 7.2) at 37 °C. I Degradation ratio of the porous Gelma hydrogel. Data was presented as the mean ± SD of three number of replicates. ∗∗p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗p < 0.0001
Fig. 2
Fig. 2
Identification of 3D-Exo released from disparate number of ADSCs (Exo 2, Exo 4 and Exo 6). A Particle size distribution measured using nanoparticle tracking analysis. B Morphological characterization via transmission electron microscopy. Scale bar: 100 nm. C Protein markers of 3D-Exo and their corresponding parental ADSCs extract quantified by western blotting
Fig. 3
Fig. 3
In vitro functional studies showing distinct effects of 3D-Exo on the proliferation, apoptosis and chondrogenesis of microtia chondrocytes. A Representative images of cell apoptosis analysis and B a quantified comparison of cell early/late apoptosis among groups of PBS control, Exo 2, Exo 4 and Exo 6. C Cell proliferation assessed by the Cell Counting Kit-8 at the specific time points after exposure to PBS, Exo 2, Exo 4 and Exo 6. D Quantitative RT-PCR analysis showed regulation of genes at 48 h post-treatment associated with anti-apoptosis (Survivin, Bcl-2), proliferation (PCNA, FGF-2), as well as chondrogenic differentiation and ear cartilage matrix proteins synthesis (TGF-β1, COL2A1, ACAN, ELN, SOX9 and COMP). The genes (COL1A1 and MMP 9) unfavorable for the synthesis of hyaline cartilage and elastic cartilage were also assessed. Data was presented as the mean ± SD of three number of replicates. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001
Fig. 4
Fig. 4
In vitro cellular uptake assay by confocal microscopy imaging demonstrated rapid uptake of 3D-Exo (red). Merged images of CFDA SE-labelled microtia chondrocyte (green) and PKH-26-labelled exosomes (red) at high magnification at 12 h revealed localization of exosomes in the cytoplasm. Scale bar: 20 μm
Fig. 5
Fig. 5
Small RNA sequencing of 3D-Exo. A Heat map showing the distinct protein profiles of the Exo 2, Exo 4, and Exo 6. Code from blue (-2 log2 normalized expression) to red (+ 2 log2 normalized expression) indicates RNA expression levels. B Volcano plot showing the number of upregulated and downregulated genes in different comparison between two types of 3D-Exo. C Venn diagram of unique and shared mRNAs in Exo 2, Exo 4, and Exo 6. D Normalized chondrogenesis-related miRNA expression levels
Fig. 6
Fig. 6
Small RNA sequencing of 3D-Exo revealed the potential mechanism on proliferation, apoptosis and new cartilage regeneration of microtia chondrocytes. A Kyoto Encyclopedia of Genes and Genomes enrichment analysis for the upregulated pathways corresponding to the comparisons. B Gene ontology analysis for the upregulated biological processes corresponding to the comparisons
Fig. 7
Fig. 7
3D-Exo activated the PTEN/P13K/AKT/mTOR signaling pathway by delivering exosomal hsa-miR-23a-3p. A The 293 T cells were transfected with luciferase reporter plasmids WT or MUT 3ʹ-UTR of PTEN and hsa-miR-23a-3p mimics/nc, and luciferase activity was detected by Dual-Luciferase Reporter Assay System. B Microtia chondrocytes were treated with 3D-Exo for 48 h, then the expression of hsa-miR-23a-3p in microtia chondrocytes was measured by qPCR. C Western blotting analysis was used for quantifying the protein levels of PTEN, P-PI3K/PI3K, P-AKT/ AKT, and mTOR in microtia chondrocytes. Data were presented as mean ± SD of three number of replicates. ***P < 0.001, ****P < 0.0001, ns: no significance
Fig. 8
Fig. 8
The hsa-miR-23a-3p overexpressing or silencing promoted or attenuated the effect of 3D-Exo on microtia chondrocytes. (A) Microtia chondrocytes were treated with hsa-miR-23a-3p mimics/inhibitor or their negative control (nc) for 24 h, and the expression of miR-23a-3p was measured by qPCR. (B) Cell proliferation assessed by the Cell Counting Kit-8 after exposure to hsa-miR-23a-3p mimics/inhibitor or their negative control (nc). (C) Representative images of cell apoptosis analysis and a quantified comparison of cell early/late apoptosis between the treatment of mimics and nc, or between the treatment of inhibitor and nc. (D) Quantitative RT-PCR analysis showed regulation of genes at 48h post-treatment associated with anti-apoptosis (Survivin, Bcl-2), proliferation (PCNA, FGF-2), as well as chondrogenic differentiation and ear cartilage matrix proteins synthesis (TGF-β1, COL2A1, ACAN, ELN, SOX9 and COMP). The genes (COL1A1 and MMP 9) unfavorable for the synthesis of hyaline cartilage and elastic cartilage were also assessed. After exposure to hsa-miR-23a-3p mimics/nc (E), or inhibitor/nc (F), western blotting analysis was used for quantifying the protein levels of PTEN, P-PI3K/PI3K, P-AKT/ AKT, mTOR and cartilage matrix (i.e. elastin, collagen II, SOX9 and collagen I) in microtia chondrocytes. Data was presented as the mean ±SD of three number of replicates. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001, ns: no significance
Fig. 9
Fig. 9
The design of Engineered-Exo. A Representative images showing the live and dead ADSCs (Parental ADSCs, agomir-23a-3p ADSCs and agomir-nc ADSCs) within porous Gelma hydrogel, and the rate of live cell. Scale bar: 500 μm. Green: live ADSCs. Red: dead ADSCs. B Quantitative RT-PCR analysis showed the level of hsa-mir-23a-3p within three types of ADSCs at 1 day and 10 day after transfection. C Protein markers of Engineered-Exo, Exo 4 and their corresponding ADSCs extract quantified by western blotting. D Quantitative RT-PCR analysis showed the level of hsa-mir-23a-3p within microtia chondrocytes at 24 and 48 h post-treatment. E Western blotting analysis was used for quantifying the protein levels of PTEN, P-PI3K/PI3K, P-AKT/ AKT and mTOR in microtia chondrocytes at 72 h post-treatment. F Quantitative RT-PCR analysis showed the mRNA level of COL2A1, ACAN, ELN, SOX9 and COL1A1 within microtia chondrocytes at 48 h post-treatment. Data was presented as the mean ± SD of three number of replicates. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001, ns: no significance
Fig. 10
Fig. 10
Engineered-Exo promoted the survival, proliferation and new cartilage formation of 3D-cultured tissue-engineered cartilage. (A) Cellular uptake assay by confocal microscopy imaging demonstrated rapid uptake of Engineered-Exo (red) at the different time points. (B) Representative images showing the live and dead microtia chondrocytes seeded on Gelma hydrogel, and (C) the comparison for the rate of live cell between Engineered-Exo and PBS control. Scale bar: 500 μm. Green: live microtia chondrocytes. Red: dead microtia chondrocytes. (D) Cell proliferation assessed by the Cell Counting Kit-8 at the specific time points after the exposure to Engineered-Exo or PBS. (E) Quantitative RT-PCR analysis showed regulation of genes associated with anti-apoptosis (PTEN, Survivin, Bcl-2), proliferation (PTEN, PCNA, FGF-2), as well as chondrogenic differentiation and ear cartilage matrix proteins synthesis (PTEN, TGF-β1, COL2A1, ACAN, ELN, SOX9 and COMP). The genes (COL1A1 and MMP 9) unfavorable for the synthesis of hyaline cartilage and elastic cartilage were also assessed. In particular, the tendency of expression of several chondrogenesis-related genes (COL2A1, ACAN, ELN, SOX9 and COL1A1) was described. (F) The protein levels of elastin, collagen II, and SOX9 in microtia chondrocytes were analyzed by western blotting. Data was presented as the mean ±SD of three number of replicates. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001
Fig. 11
Fig. 11
The effects of a single dose of Engineered-Exo on tissue-engineered cartilage regeneration. A Gross observation at 6 weeks after implantation. B Quantitative RT-PCR analysis showed regulation of genes at 6 weeks after implantation associated with ear cartilage matrix proteins synthesis. C The tissue-engineered cartilage was histologically evaluated via staining of hematoxylin and eosin (HE), Safranin O, Alcian blue and Toluidine blue. Scale bars: 100 μm. Data was presented as the mean ± SD of three number of replicates. ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001
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