Genome-Wide CRISPR/Cas9-Based Screening for Deubiquitinase Subfamily Identifies Ubiquitin-Specific Protease 11 as a Novel Regulator of Osteogenic Differentiation

The osteoblast differentiation capacity of mesenchymal stem cells must be tightly regulated, as inadequate bone mineralization can lead to osteoporosis, and excess bone formation can cause the heterotopic ossification of soft tissues. The balanced protein level of Msh homeobox 1 (MSX1) is critical during normal osteogenesis. To understand the factors that prevent MSX1 protein degradation, the identification of deubiquitinating enzymes (DUBs) for MSX1 is essential. In this study, we performed loss-of-function-based screening for DUBs regulating MSX1 protein levels using the CRISPR/Cas9 system. We identified ubiquitin-specific protease 11 (USP11) as a protein regulator of MSX1 and further demonstrated that USP11 interacts and prevents MSX1 protein degradation by its deubiquitinating activity. Overexpression of USP11 enhanced the expression of several osteogenic transcriptional factors in human mesenchymal stem cells (hMSCs). Additionally, differentiation studies revealed reduced calcification and alkaline phosphatase activity in USP11-depleted cells, while overexpression of USP11 enhanced the differentiation potential of hMSCs. These results indicate the novel role of USP11 during osteogenic differentiation and suggest USP11 as a potential target for bone regeneration.

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MicroRNA treatment modulates osteogenic differentiation potential of mesenchymal stem cells derived from human chorion and placenta

Scientific Reports ◽

10.1038/s41598-021-87298-5 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Kulisara Marupanthorn ◽

Chairat Tantrawatpan ◽

Pakpoom Kheolamai ◽

Duangrat Tantikanlayaporn ◽

Sirikul Manochantr

Keyword(s):

Gene Expression ◽

Stem Cells ◽

Bone Marrow ◽

Mesenchymal Stem Cells ◽

Transcription Factor ◽

Osteogenic Differentiation ◽

Differentiation Potential ◽

Osteogenic Gene Expression ◽

Osteogenic Gene

AbstractMesenchymal stem cells (MSCs) are important in regenerative medicine because of their potential for multi-differentiation. Bone marrow, chorion and placenta have all been suggested as potential sources for clinical application. However, the osteogenic differentiation potential of MSCs derived from chorion or placenta is not very efficient. Bone morphogenetic protein-2 (BMP-2) plays an important role in bone development. Its effect on osteogenic augmentation has been addressed in several studies. Recent studies have also shown a relationship between miRNAs and osteogenesis. We hypothesized that miRNAs targeted to Runt-related transcription factor 2 (Runx-2), a major transcription factor of osteogenesis, are responsible for regulating the differentiation of MSCs into osteoblasts. This study examines the effect of BMP-2 on the osteogenic differentiation of MSCs isolated from chorion and placenta in comparison to bone marrow-derived MSCs and investigates the role of miRNAs in the osteogenic differentiation of MSCs from these sources. MSCs were isolated from human bone marrow, chorion and placenta. The osteogenic differentiation potential after BMP-2 treatment was examined using ALP staining, ALP activity assay, and osteogenic gene expression. Candidate miRNAs were selected and their expression levels during osteoblastic differentiation were examined using real-time RT-PCR. The role of these miRNAs in osteogenesis was investigated by transfection with specific miRNA inhibitors. The level of osteogenic differentiation was monitored after anti-miRNA treatment. MSCs isolated from chorion and placenta exhibited self-renewal capacity and multi-lineage differentiation potential similar to MSCs isolated from bone marrow. BMP-2 treated MSCs showed higher ALP levels and osteogenic gene expression compared to untreated MSCs. All investigated miRNAs (miR-31, miR-106a and miR148) were consistently downregulated during the process of osteogenic differentiation. After treatment with miRNA inhibitors, ALP activity and osteogenic gene expression increased over the time of osteogenic differentiation. BMP-2 has a positive effect on osteogenic differentiation of chorion- and placenta-derived MSCs. The inhibition of specific miRNAs enhanced the osteogenic differentiation capacity of various MSCs in culture and this strategy might be used to promote bone regeneration. However, further in vivo experiments are required to assess the validity of this approach.

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Homeobox C10 inhibits the osteogenic differentiation potential of mesenchymal stem cells

Connective Tissue Research ◽

10.1080/03008207.2017.1341496 ◽

2017 ◽

pp. 1-11 ◽

Cited By ~ 3

Author(s):

Guoqing Li ◽

Nannan Han ◽

Haoqing Yang ◽

Liping Wang ◽

Xiao Lin ◽

...

Keyword(s):

Stem Cells ◽

Mesenchymal Stem Cells ◽

Osteogenic Differentiation ◽

Differentiation Potential

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Osteogenic differentiation potential and marker gene expression of different porcine bone marrow mesenchymal stem cell subpopulations selected in different basal media

10.1101/2020.04.27.063230 ◽

2020 ◽

Author(s):

Sangeetha Kannan ◽

Jyotirmoy Ghosh ◽

Sujoy K. Dhara

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Mesenchymal Stem Cells ◽

Osteogenic Differentiation ◽

Marker Gene ◽

Microscopical Examination ◽

Alizarin Red ◽

Differentiation Potential ◽

Basal Media ◽

Selection Of

AbstractMultipotent porcine mesenchymal stem cells (pMSC) are indispensable for research and therapeutic use. Derivation and culture media might affect the selection of MSC subpopulation and thus the differentiation potential of cells. In this study we evaluated the effects of αMEM, aDMEM, M199, αMEM/M199, aDMEM/M199 and αMEM/aDMEM media on porcine bone marrow MSC derivation; pre-differentiation expression of ALP, COL1A1, SPP1 and BGLAP osteogenic marker genes at passage 5 and 10 pMSC; and differentiation potential of passage 5 pMSC. Morphological changes and matrix formation in osteogenic cells were evaluated by microscopical examination and calcium deposit in osteocytes was confirmed by Alizarin Red S staining. Results indicated media independent selection of different bone marrow MSC subpopulations with different surface marker gene expressions. Many pMSC subpopulations in different media had CD14+ expressing cells. We also observed basal media dependent changes in osteogenic markers expression and differentiation potential of pMSC. The αMEM/aDMEM media grown pMSC showed best osteogenic differentiation potential. We thus recommended the testing of αMEM/aDMEM mixed media in other species for pre-differentiation MSC culture that are intended for better osteogenic differentiation.SummaryPre-differentiation basal media influence osteogenic differentiation potential of mesenchymal stem cells (MSC). Among the tested media, αMEM/aDMEM was the best for pre-differentiation porcine MSC culture intending to use in osteogenesis.

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Differential Expression of MicroRNA-3148 in Patients with Osteoporosis and Its Impacts on the Osteogenic Differentiation of Rat Bone Marrow Mesenchymal Stem Cells

Journal of Biomaterials and Tissue Engineering ◽

10.1166/jbt.2021.2635 ◽

2021 ◽

Vol 11 (5) ◽

pp. 957-962

Author(s):

Ainiwaerjiang Damaola ◽

Maerdan Aierken ◽

Mieralimu Muertizha ◽

Abudouaini Abudoureheman ◽

Haishan Lin ◽

...

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Mesenchymal Stem Cells ◽

Osteogenic Differentiation ◽

Serum Samples ◽

Alizarin Red ◽

Differentiation Potential ◽

Qrt Pcr ◽

Marrow Mesenchymal Stem Cells ◽

Rat Bone

We aimed to explore the effects of rat bone marrow mesenchymal stem cells (BMSCs) on osteogenic differentiation via analyzing miR-3148 expression in patients with osteoporosis. Realtime quantitative PCR was conducted for assessing microRNA-3148 expression. BMSCs from SD rats were transfected with microRNA-3148 mimics and microRNA-3148 inhibitor via liposomal trans-fection method utilizing Lipo2000, followed by analysis of microRNA-3148 level. After 10-days of osteogenic differentiation induction, alkaline phosphatase (ALP) staining and alizarin red (ARS) staining were done to investigate the osteogenic differentiation potential. Simultaneously, qRT-PCR measured the expression of osteogenesis marker genes (BMP and Runx2) in each group. qRT-PCR analysis revealed a high expression of miR-3148 in the bone tissue and the serum samples from patients with osteoporosis in comparison with healthy individuals. In addition, miRNA-3148 mimics could retard the osteogenic differentiation of BMSCs, while microRNA-3148 inhibitor could prompt the procedure. MicroRNA-3148 was highly expressed in the skeletal tissues and the serum samples from patients with osteoporosis and it could restrain the differentiation of BMSCs into osteoblasts, suggesting that it might be a novel therapeutic target for treating osteoporosis.

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Different osteogenic differentiation potential of mesenchymal stem cells on three different polymeric substrates

Gene ◽

10.1016/j.gene.2020.144534 ◽

2020 ◽

Vol 740 ◽

pp. 144534 ◽

Cited By ~ 5

Author(s):

Mohammad Foad Abazari ◽

Zahrasadat Hosseini ◽

Shohreh Zare Karizi ◽

Sara Norouzi ◽

Mojdeh Amini Faskhoudi ◽

...

Keyword(s):

Stem Cells ◽

Mesenchymal Stem Cells ◽

Osteogenic Differentiation ◽

Differentiation Potential ◽

Polymeric Substrates

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CircRNA-vgll3 promotes osteogenic differentiation of adipose-derived mesenchymal stem cells via modulating miRNA-dependent integrin α5 expression

Cell Death and Differentiation ◽

10.1038/s41418-020-0600-6 ◽

2020 ◽

Vol 28 (1) ◽

pp. 283-302

Author(s):

Dandan Zhang ◽

Ni Ni ◽

Yuyao Wang ◽

Zhimin Tang ◽

Huiqin Gao ◽

...

Keyword(s):

Stem Cells ◽

Mesenchymal Stem Cells ◽

Regenerative Medicine ◽

Osteogenic Differentiation ◽

Progenitor Cell ◽

Bone Repair ◽

Circular Rnas ◽

Mineral Density ◽

Differentiation Potential ◽

Integrin Α5

AbstractAdipose-derived mesenchymal stem cells (ADSCs) are promising candidate for regenerative medicine to repair non-healing bone defects due to their high and easy availability. However, the limited osteogenic differentiation potential greatly hinders the clinical application of ADSCs in bone repair. Accumulating evidences demonstrate that circular RNAs (circRNAs) are involved in stem/progenitor cell fate determination, but their specific role in stem/progenitor cell osteogenesis, remains mostly undescribed. Here, we show that circRNA-vgll3 originating from the vgll3 locus markedly enhances osteogenic differentiation of ADSCs; nevertheless, silencing of circRNA-vgll3 dramatically attenuates ADSC osteogenesis. Furthermore, we validate that circRNA-vgll3 functions in ADSC osteogenesis through a circRNA-vgll3/miR-326-5p/integrin α5 (Itga5) pathway. Itga5 promotes ADSC osteogenic differentiation and miR-326-5p suppresses Itga5 translation. CircRNA-vgll3 directly sequesters miR-326-5p in the cytoplasm and inhibits its activity to promote osteogenic differentiation. Moreover, the therapeutic potential of circRNA-vgll3-modified ADSCs with calcium phosphate cement (CPC) scaffolds was systematically evaluated in a critical-sized defect model in rats. Our results demonstrate that circRNA-vgll3 markedly enhances new bone formation with upregulated bone mineral density, bone volume/tissue volume, trabeculae number, and increased new bone generation. This study reveals the important role of circRNA-vgll3 during new bone biogenesis. Thus, circRNA-vgll3 engineered ADSCs may be effective potential therapeutic targets for bone regenerative medicine.

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Osteogenic differentiation potential of porcine bone marrow mesenchymal stem cell subpopulations selected in different basal media

Biology Open ◽

10.1242/bio.053280 ◽

2020 ◽

Vol 9 (10) ◽

pp. bio053280

Author(s):

Sangeetha Kannan ◽

Jyotirmoy Ghosh ◽

Sujoy K. Dhara

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Mesenchymal Stem Cells ◽

Osteogenic Differentiation ◽

Alizarin Red ◽

Differentiation Potential ◽

Osteogenic Lineage ◽

Osteogenic Markers ◽

Basal Media ◽

Selection Of

ABSTRACTMultipotent porcine mesenchymal stem cells (pMSC) are invaluable for research and therapeutic use in regenerative medicine. Media used for derivation and expansion of pMSC may play an important role for the selection of MSC subpopulation at an early stage and thereby, the specific basal medium may also affect differentiation potential of these cells. The present study was undertaken to evaluate the effects of αMEM, aDMEM, M199, αMEM/M199, aDMEM/M199 and αMEM/aDMEM media on (1) porcine bone marrow MSC derivation; (2) expression of number of osteogenic markers (ALP, COL1A1, SPP1 and BGLAP) at 5th and 10th passage in pMSC before differentiation; and (3) differentiation of pMSC (at 5th passage) to osteogenic lineage. Morphological changes and matrix formation in osteogenic cells were evaluated by microscopic examination. Calcium deposits in osteocytes were confirmed by Alizarin Red S staining. Based on expression of different markers, it was evident that selection of bone marrow pMSC subpopulations was independent of basal media used. However, the differentiation of those pMSCs, specifically to osteogenic lineage, was dependent on the medium used for expansion of pMSC at the pre-differentiation stage. We demonstrated here that the pMSC grown in combined αMEM/aDMEM (1:1) medium expressed number of osteogenic markers and these pMSC underwent osteogenic differentiation most efficiently, in comparison to porcine mesenchymal stem cells grown in other media. In conclusion, osteogenic differentiation potential of pMSC maintained in αMEM/aDMEM medium was observed significantly higher compared to cells cultivated in other media and therefore, the combined medium αMEM/aDMEM (1:1) may preferentially be used for expansion of pMSC, if needed for osteogenic differentiation.

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Biological Characteristics and Osteogenic Differentiation of Ovine Bone Marrow Derived Mesenchymal Stem Cells Stimulated with FGF-2 and BMP-2

International Journal of Molecular Sciences ◽

10.3390/ijms21249726 ◽

2020 ◽

Vol 21 (24) ◽

pp. 9726

Author(s):

Sandra Gromolak ◽

Agnieszka Krawczenko ◽

Agnieszka Antończyk ◽

Krzysztof Buczak ◽

Zdzisław Kiełbowicz ◽

...

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Mesenchymal Stem Cells ◽

Osteogenic Differentiation ◽

Collagen Type ◽

Collagen Type I ◽

Biological Characteristics ◽

Immunofluorescent Staining ◽

Type I ◽

Differentiation Potential

Cell-based therapies using mesenchymal stem cells (MSCs) are a promising tool in bone tissue engineering. Bone regeneration with MSCs involves a series of molecular processes leading to the activation of the osteoinductive cascade supported by bioactive factors, including fibroblast growth factor-2 (FGF-2) and bone morphogenetic protein-2 (BMP-2). In this study, we examined the biological characteristics and osteogenic differentiation potential of sheep bone marrow MSCs (BM-MSCs) treated with 20 ng/mL of FGF-2 and 100 ng/mL BMP-2 in vitro. The biological properties of osteogenic-induced BM-MSCs were investigated by assessing their morphology, proliferation, phenotype, and cytokine secretory profile. The osteogenic differentiation was characterized by Alizarin Red S staining, immunofluorescent staining of osteocalcin and collagen type I, and expression levels of genetic markers of osteogenesis. The results demonstrated that BM-MSCs treated with FGF-2 and BMP-2 maintained their primary MSC properties and improved their osteogenic differentiation capacity, as confirmed by increased expression of osteocalcin and collagen type I and upregulation of osteogenic-related gene markers BMP-2, Runx2, osterix, collagen type I, osteocalcin, and osteopontin. Furthermore, sheep BM-MSCs produced a variety of bioactive factors involved in osteogenesis, and supplementation of the culture medium with FGF-2 and BMP-2 affected the secretome profile of the cells. The results suggest that sheep osteogenic-induced BM-MSCs may be used as a cellular therapy to study bone repair in the preclinical large animal model.

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