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Bonilauri B, Ribeiro AL, Spangenberg L, Dallagiovanna B. Unveiling Polysomal Long Non-Coding RNA Expression on the First Day of Adipogenesis and Osteogenesis in Human Adipose-Derived Stem Cells. Int J Mol Sci 2024; 25:2013. [PMID: 38396700 PMCID: PMC10888724 DOI: 10.3390/ijms25042013] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/12/2023] [Revised: 01/06/2024] [Accepted: 01/11/2024] [Indexed: 02/25/2024] Open
Abstract
Understanding the intricate molecular mechanisms governing the fate of human adipose-derived stem cells (hASCs) is essential for elucidating the delicate balance between adipogenic and osteogenic differentiation in both healthy and pathological conditions. Long non-coding RNAs (lncRNAs) have emerged as key regulators involved in lineage commitment and differentiation of stem cells, operating at various levels of gene regulation, including transcriptional, post-transcriptional, and post-translational processes. To gain deeper insights into the role of lncRNAs' in hASCs' differentiation, we conducted a comprehensive analysis of the lncRNA transcriptome (RNA-seq) and translatome (polysomal-RNA-seq) during a 24 h period of adipogenesis and osteogenesis. Our findings revealed distinct expression patterns between the transcriptome and translatome during both differentiation processes, highlighting 90 lncRNAs that are exclusively regulated in the polysomal fraction. These findings underscore the significance of investigating lncRNAs associated with ribosomes, considering their unique expression patterns and potential mechanisms of action, such as translational regulation and potential coding capacity for microproteins. Additionally, we identified specific lncRNA gene expression programs associated with adipogenesis and osteogenesis during the early stages of cell differentiation. By shedding light on the expression and potential functions of these polysome-associated lncRNAs, we aim to deepen our understanding of their involvement in the regulation of adipogenic and osteogenic differentiation, ultimately paving the way for novel therapeutic strategies and insights into regenerative medicine.
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Affiliation(s)
- Bernardo Bonilauri
- Stem Cell Basic Biology Laboratory (LABCET), Carlos Chagas Institute—Fiocruz/PR, Curitiba 81350-010, PR, Brazil;
- Stanford Cardiovascular Institute, Stanford University School of Medicine, Stanford, CA 94305, USA
| | - Annanda Lyra Ribeiro
- Stem Cell Basic Biology Laboratory (LABCET), Carlos Chagas Institute—Fiocruz/PR, Curitiba 81350-010, PR, Brazil;
| | - Lucía Spangenberg
- Bioinformatics Unit, Institut Pasteur de Montevideo, Montevideo 11400, Uruguay;
| | - Bruno Dallagiovanna
- Stem Cell Basic Biology Laboratory (LABCET), Carlos Chagas Institute—Fiocruz/PR, Curitiba 81350-010, PR, Brazil;
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Carnieri MV, Garcia DDF, Voltolini R, Volpato N, Mafra M, Bernardelli EA, Stimamiglio MA, Rebelatto CK, Correa A, Berti LF, Marcon BH. Cytocompatible and osteoconductive silicon oxycarbide glass scaffolds 3D printed by DLP: a potential material for bone tissue regeneration. Front Bioeng Biotechnol 2024; 11:1297327. [PMID: 38239914 PMCID: PMC10794595 DOI: 10.3389/fbioe.2023.1297327] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/19/2023] [Accepted: 12/12/2023] [Indexed: 01/22/2024] Open
Abstract
Bone lesions affect individuals of different age groups, compromising their daily activities and potentially leading to prolonged morbidity. Over the years, new compositions and manufacturing technologies were developed to offer customized solutions to replace injured tissue and stimulate tissue regeneration. This work used digital light processing (DPL) technology for three-dimensional (3D) printing of porous structures using pre-ceramic polymer, followed by pyrolysis to obtain SiOC vitreous scaffolds. The SiOC scaffolds produced had an amorphous structure (compatible with glass) with an average porosity of 72.69% ± 0.99, an average hardness of 935.1 ± 71.0 HV, and an average maximum flexural stress of 7.8 ± 1.0 MPa, similar to cancellous bone tissue. The scaffolds were not cytotoxic and allowed adult stem cell adhesion, growth, and expansion. After treatment with osteoinductive medium, adult stem cells in the SiOC scaffolds differentiated to osteoblasts, assuming a tissue-like structure, with organization in multiple layers and production of a dense fibrous matrix rich in hydroxyapatite. The in vitro analyses supported the hypothesis that the SiOC scaffolds produced in this work were suitable for use as a bone substitute for treating critically sized lesions, with the potential to stimulate the gradual process of regeneration of the native tissue. The data obtained stimulate the continuity of studies with the SiOC scaffolds developed in this work, paving the way for evaluating safety and biological activity in vivo.
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Affiliation(s)
- Matheus Versão Carnieri
- Department of Mechanical Engineering, Postgraduate Program in Mechanical and Materials Engineering, Universidade Tecnológica Federal Do Parana, Curitiba, Brazil
| | - Daniele de Freitas Garcia
- Laboratory of Basic Biology of Stem Cells (LABCET), Carlos Chagas Institute—FIOCRUZ-PR, Curitiba, Brazil
| | - Rafael Voltolini
- Department of Mechanical Engineering, Postgraduate Program in Mechanical and Materials Engineering, Universidade Tecnológica Federal Do Parana, Curitiba, Brazil
| | - Neri Volpato
- Department of Mechanical Engineering, Postgraduate Program in Mechanical and Materials Engineering, Universidade Tecnológica Federal Do Parana, Curitiba, Brazil
| | - Marcio Mafra
- Department of Mechanical Engineering, Postgraduate Program in Mechanical and Materials Engineering, Universidade Tecnológica Federal Do Parana, Curitiba, Brazil
| | - Euclides Alexandre Bernardelli
- Department of Mechanical Engineering, Postgraduate Program in Mechanical and Materials Engineering, Universidade Tecnológica Federal Do Parana, Curitiba, Brazil
| | - Marco Augusto Stimamiglio
- Laboratory of Basic Biology of Stem Cells (LABCET), Carlos Chagas Institute—FIOCRUZ-PR, Curitiba, Brazil
| | | | - Alejandro Correa
- Laboratory of Basic Biology of Stem Cells (LABCET), Carlos Chagas Institute—FIOCRUZ-PR, Curitiba, Brazil
| | - Lucas Freitas Berti
- Department of Mechanical Engineering, Postgraduate Program in Mechanical and Materials Engineering, Universidade Tecnológica Federal Do Parana, Curitiba, Brazil
| | - Bruna Hilzendeger Marcon
- Laboratory of Basic Biology of Stem Cells (LABCET), Carlos Chagas Institute—FIOCRUZ-PR, Curitiba, Brazil
- Confocal and Eletronic Microscopy Facility (RPT07C), Carlos Chagas Institute—FIOCRUZ-PR, Curitiba, Brazil
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Nogoceke R, Josino R, Robert AW, Stimamiglio MA. Evaluation of a Peptide Hydrogel as a Chondro-Instructive Three-Dimensional Microenvironment. Polymers (Basel) 2023; 15:4630. [PMID: 38139882 PMCID: PMC10747086 DOI: 10.3390/polym15244630] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/20/2023] [Revised: 11/16/2023] [Accepted: 11/19/2023] [Indexed: 12/24/2023] Open
Abstract
Articular cartilage injuries are inherently irreversible, even with the advancement in current therapeutic options. Alternative approaches, such as the use of mesenchymal stem/stromal cells (MSCs) and tissue engineering techniques, have gained prominence. MSCs represent an ideal source of cells due to their low immunogenicity, paracrine activity, and ability to differentiate. Among biomaterials, self-assembling peptide hydrogels (SAPH) are interesting given their characteristics such as good biocompatibility and tunable properties. Herein we associate human adipose-derived stem cells (hASCs) with a commercial SAPH, Puramatrix™, to evaluate how this three-dimensional microenvironment affects cell behavior and its ability to undergo chondrogenic differentiation. We demonstrate that the Puramatrix™ hydrogel comprises a highly porous matrix permissible for hASC adhesion and in vitro expansion. The morphology and cell growth dynamics of hASCs were affected when cultured on the hydrogel but had minimal alteration in their immunophenotype. Interestingly, hASCs spontaneously formed cell aggregates throughout culturing. Analysis of glycosaminoglycan production and gene expression revealed a noteworthy and donor-dependent trend suggesting that Puramatrix™ hydrogel may have a natural capacity to support the chondrogenic differentiation of hASCs. Altogether, the results provide a more comprehensive understanding of the potential applications and limitations of the Puramatrix™ hydrogel in developing functional cartilage tissue constructs.
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Affiliation(s)
| | | | - Anny Waloski Robert
- Stem Cells Basic Biology Laboratory, Instituto Carlos Chagas—ICC-FIOCRUZ/PR, Rua Professor Algacyr Munhoz Mader, 3775, Curitiba 81350-010, Brazil; (R.N.); (R.J.)
| | - Marco Augusto Stimamiglio
- Stem Cells Basic Biology Laboratory, Instituto Carlos Chagas—ICC-FIOCRUZ/PR, Rua Professor Algacyr Munhoz Mader, 3775, Curitiba 81350-010, Brazil; (R.N.); (R.J.)
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Proteogenomic Analysis Reveals Proteins Involved in the First Step of Adipogenesis in Human Adipose-Derived Stem Cells. Stem Cells Int 2021; 2021:3168428. [PMID: 34956370 PMCID: PMC8702357 DOI: 10.1155/2021/3168428] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/06/2021] [Accepted: 11/22/2021] [Indexed: 12/13/2022] Open
Abstract
Background Obesity is characterized as a disease that directly affects the whole-body metabolism and is associated with excess fat mass and several related comorbidities. Dynamics of adipocyte hypertrophy and hyperplasia play an important role in health and disease, especially in obesity. Human adipose-derived stem cells (hASC) represent an important source for understanding the entire adipogenic differentiation process. However, little is known about the triggering step of adipogenesis in hASC. Here, we performed a proteogenomic approach for understanding the protein abundance alterations during the initiation of the adipogenic differentiation process. Methods hASC were isolated from adipose tissue of three donors and were then characterized and expanded. Cells were cultured for 24 hours in adipogenic differentiation medium followed by protein extraction. We used shotgun proteomics to compare the proteomic profile of 24 h-adipogenic, differentiated, and undifferentiated hASC. We also used our previous next-generation sequencing data (RNA-seq) of the total and polysomal mRNA fractions of hASC to study posttranscriptional regulation during the initial steps of adipogenesis. Results We identified 3420 proteins out of 48,336 peptides, of which 92 proteins were exclusively identified in undifferentiated hASC and 53 proteins were exclusively found in 24 h-differentiated cells. Using a stringent criterion, we identified 33 differentially abundant proteins when comparing 24 h-differentiated and undifferentiated hASC (14 upregulated and 19 downregulated, respectively). Among the upregulated proteins, we shortlisted several adipogenesis-related proteins. A combined analysis of the proteome and the transcriptome allowed the identification of positive correlation coefficients between proteins and mRNAs. Conclusions These results demonstrate a specific proteome profile related to adipogenesis at the beginning (24 hours) of the differentiation process in hASC, which advances the understanding of human adipogenesis and obesity. Adipogenic differentiation is finely regulated at the transcriptional, posttranscriptional, and posttranslational levels.
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Bonilauri B, Holetz FB, Dallagiovanna B. Long Non-Coding RNAs Associated with Ribosomes in Human Adipose-Derived Stem Cells: From RNAs to Microproteins. Biomolecules 2021; 11:1673. [PMID: 34827671 PMCID: PMC8615451 DOI: 10.3390/biom11111673] [Citation(s) in RCA: 6] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/06/2021] [Revised: 10/15/2021] [Accepted: 10/25/2021] [Indexed: 12/12/2022] Open
Abstract
Ribosome profiling reveals the translational dynamics of mRNAs by capturing a ribosomal footprint snapshot. Growing evidence shows that several long non-coding RNAs (lncRNAs) contain small open reading frames (smORFs) that are translated into functional peptides. The difficulty in identifying bona-fide translated smORFs is a constant challenge in experimental and bioinformatics fields due to their unconventional characteristics. This motivated us to isolate human adipose-derived stem cells (hASC) from adipose tissue and perform a ribosome profiling followed by bioinformatics analysis of transcriptome, translatome, and ribosome-protected fragments of lncRNAs. Here, we demonstrated that 222 lncRNAs were associated with the translational machinery in hASC, including the already demonstrated lncRNAs coding microproteins. The ribosomal occupancy of some transcripts was consistent with the translation of smORFs. In conclusion, we were able to identify a subset of 15 lncRNAs containing 35 smORFs that likely encode functional microproteins, including four previously demonstrated smORF-derived microproteins, suggesting a possible dual role of these lncRNAs in hASC self-renewal.
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Affiliation(s)
- Bernardo Bonilauri
- Laboratory of Basic Biology of Stem Cells (LABCET), Carlos Chagas Institute-Fiocruz-Paraná, Curitiba 81350-010, Brazil;
| | - Fabiola Barbieri Holetz
- Laboratory of Gene Expression Regulation (LABREG), Carlos Chagas Institute-Fiocruz-Paraná, Curitiba 81350-010, Brazil;
| | - Bruno Dallagiovanna
- Laboratory of Basic Biology of Stem Cells (LABCET), Carlos Chagas Institute-Fiocruz-Paraná, Curitiba 81350-010, Brazil;
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Wang C, Dong L, Wang Y, Jiang Z, Zhang J, Yang G. Bioinformatics Analysis Identified miR-584-5p and Key miRNA-mRNA Networks Involved in the Osteogenic Differentiation of Human Periodontal Ligament Stem Cells. Front Genet 2021; 12:750827. [PMID: 34646313 PMCID: PMC8503254 DOI: 10.3389/fgene.2021.750827] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/31/2021] [Accepted: 08/30/2021] [Indexed: 12/29/2022] Open
Abstract
Human periodontal ligament cells (PDLCs) play an important role in periodontal tissue stabilization and function. In the process of osteogenic differentiation of PDLSCs, the regulation of molecular signal pathways are complicated. In this study, the sequencing results of three datasets on GEO were used to comprehensively analyze the miRNA-mRNA network during the osteogenic differentiation of PDLSCs. Using the GSE99958 and GSE159507, a total of 114 common differentially expressed genes (DEGs) were identified, including 62 up-regulated genes and 52 down-regulated genes. GO enrichment analysis was performed. The up-regulated 10 hub genes and down-regulated 10 hub genes were screened out by protein-protein interaction network (PPI) analysis and STRING in Cytoscape. Similarly, differentially expressed miRNAs (DEMs) were selected by limma package from GSE159508. Then, using the miRwalk website, we further selected 11 miRNAs from 16 DEMs that may have a negative regulatory relationship with hub genes. In vitro RT-PCR verification revealed that nine DEMs and 18 hub genes showed the same trend as the RNA-seq results during the osteogenic differentiation of PDLSCs. Finally, using miR-584-5p inhibitor and mimics, it was found that miR-584-5p negatively regulates the osteogenic differentiation of PDLSCs in vitro. In summary, the present results found several potential osteogenic-related genes and identified candidate miRNA-mRNA networks for the further study of osteogenic differentiation of PDLSCs.
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Affiliation(s)
| | | | | | | | | | - Guoli Yang
- Key Laboratory of Oral Biomedical Research of Zhejiang Province, The Affiliated Hospital of Stomatology, School of Stomatology, Zhejiang University School of Medicine, Hangzhou, China
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Horinouchi CDS, Barisón MJ, Robert AW, Kuligovski C, Aguiar AM, Dallagiovanna B. Influence of donor age on the differentiation and division capacity of human adipose-derived stem cells. World J Stem Cells 2020; 12:1640-1651. [PMID: 33505605 PMCID: PMC7789122 DOI: 10.4252/wjsc.v12.i12.1640] [Citation(s) in RCA: 16] [Impact Index Per Article: 3.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 07/16/2020] [Revised: 10/09/2020] [Accepted: 11/05/2020] [Indexed: 02/06/2023] Open
Abstract
BACKGROUND Human adipose-derived stromal/stem cells (hASCs) are one of the most useful types of mesenchymal stromal/stem cells, which are adult multipotent cells with great therapeutic potential for the treatment of several diseases. However, for successful clinical application, it is critical that high-quality cells can be obtained. Diverse factors seem to be able to influence cell quality and performance, especially factors related to donors’ intrinsic characteristics, such as age. Nevertheless, there is no consensus regarding this characteristic, and there is conflicting information in the literature.
AIM To investigate the growth kinetics and differentiation potential of adipose-derived stem cells isolated from the lipoaspirates of elderly and young donors.
METHODS hASCs were harvested from liposuctioned adipose tissue obtained from female donors (aged 20-70 years). Cells were distributed into two groups according to age range: old hASCs (oASCs, ≥ 55 years, n = 9) and young hASCs (yASCs, ≤ 35 years, n = 9). For each group, immunophenotypic characterization was performed by flow cytometry. Population doubling time was assessed over seven days. For adipogenic potential evaluation, lipid deposits were assessed after 7 d, 14 d and 21 d of adipogenic induction. Osteogenic potential was verified by analyzing cell mineralization after 14 d, 21 d and 28 d of osteogenic induction. mRNA expression of PPARγ2, CEBPA and Runx2 were detected by quantitative reverse transcription polymerase chain reaction.
RESULTS hASCs were successfully obtained, cultured, and grouped according to their age: yASCs (26.33 ± 4.66 years old) and oASCs (64.78 ± 4.58 years old). After maintenance of the cells in culture, there were no differences in morphology between cells from the young and old donors. Additionally, both groups showed classical immunophenotypic characteristics of mesenchymal stem/stromal cells. The average doubling time indicated that yASCs (4.09 ± 0.94 d) did not significantly differ from oASCs (4.19 ± 1.29 d). Concerning differentiation potential, after adipogenic and osteogenic induction, yASCs and oASCs were able to differentiate to greater levels than the noninduced control cells. However, no differences were found in the differentiation efficiency of yASCs and oASCs in adipogenesis or osteogenesis. Additionally, the mRNA expression of PPARγ2, CEBPA and Runx2 were similar in yASCs and oASCs.
CONCLUSION Our findings suggest that age does not seem to significantly affect the cell division or adipogenic or osteogenic differentiation ability of adipose-derived stem cells isolated from lipoaspirates.
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Affiliation(s)
- Cintia DS Horinouchi
- Stem Cells Basic Biology Laboratory, Instituto Carlos Chagas, Curitiba 81350010, Paraná, Brazil
| | - María Julia Barisón
- Stem Cells Basic Biology Laboratory, Instituto Carlos Chagas, Curitiba 81350010, Paraná, Brazil
| | - Anny W Robert
- Stem Cells Basic Biology Laboratory, Instituto Carlos Chagas, Curitiba 81350010, Paraná, Brazil
| | - Crisciele Kuligovski
- Stem Cells Basic Biology Laboratory, Instituto Carlos Chagas, Curitiba 81350010, Paraná, Brazil
| | - Alessandra M Aguiar
- Stem Cells Basic Biology Laboratory, Instituto Carlos Chagas, Curitiba 81350010, Paraná, Brazil
| | - Bruno Dallagiovanna
- Stem Cells Basic Biology Laboratory, Instituto Carlos Chagas, Curitiba 81350010, Paraná, Brazil
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Robert AW, Marcon BH, Dallagiovanna B, Shigunov P. Adipogenesis, Osteogenesis, and Chondrogenesis of Human Mesenchymal Stem/Stromal Cells: A Comparative Transcriptome Approach. Front Cell Dev Biol 2020; 8:561. [PMID: 32733882 PMCID: PMC7362937 DOI: 10.3389/fcell.2020.00561] [Citation(s) in RCA: 88] [Impact Index Per Article: 17.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/31/2020] [Accepted: 06/12/2020] [Indexed: 12/20/2022] Open
Abstract
Adipogenesis, osteogenesis and chondrogenesis of human mesenchymal stem/stromal cells (MSC) are complex and highly regulated processes. Over the years, several studies have focused on understanding the mechanisms involved in the MSC commitment to the osteogenic, adipogenic and/or chondrogenic phenotypes. High-throughput methodologies have been used to investigate the gene expression profile during differentiation. Association of data analysis of mRNAs, microRNAs, circular RNAs and long non-coding RNAs, obtained at different time points over these processes, are important to depict the complexity of differentiation. This review will discuss the results that were highlighted in transcriptome analyses of MSC undergoing adipogenic, osteogenic and chondrogenic differentiation. The focus is to shed light on key molecules, main signaling pathways and biological processes related to different time points of adipogenesis, osteogenesis and chondrogenesis.
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Affiliation(s)
- Anny W Robert
- Instituto Carlos Chagas - Fiocruz Paraná, Curitiba, Brazil
| | - Bruna H Marcon
- Instituto Carlos Chagas - Fiocruz Paraná, Curitiba, Brazil
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