7 research outputs found
Suramin inhibits chikungunya virus replication through multiple mechanisms
No full textMolecular basis of virus replication, viral pathogenesis and antiviral strategie
654-Timely modification of microenvironment stiffness improves chondrogenesis of single cell HIMSCS
No full textPurpose (the aim of the study): Generation of de novo cartilage from cell types such as hiPSC-derived mesenchymal stromal cells (hiMSCs) are attractive due to their benefits as a stable and sustainable cell source for therapies and disease modelling. Nonetheless, the quality of neocartilage generated by hiMSCs is inferior compared to that of primary chondrocytes (Rodriguez Ruiz, Dicks et al. 2021). To improve hiMSC-chondrogenesis, unbiased characterization of modifying factors that can direct cell fate during differentiation at the single cell level is needed. The impact of microenvironment stiffness has previously been shown for differentiation to adipose and bone and here we apply the same principles (Kamperman, Henke et al. 2021). By encapsulating hiMSCs in single cell microgels of with precisely controlled microenvironment stiffness to improve chondrogenesis, and allow for the easy isolation of single cells for advanced characterization.Methods: hiMSCs were generated (Rodriguez Ruiz, Dicks et al. 2021, Ramos, Tertel et al. 2022) and encapsulated as single cells in 5% Dex-TA using two microfluidic chips (one for droplet generation and another for delayed gelation) (Kamperman, Henke et al. 2017, Kamperman, Henke et al. 2021). hiMSCs were encapsulated in microgels crosslinked to 20kPa (soft) or 75kPa (stiff). Stiffness of the microgels was managed through control of the concentration of H2O2 used in the crosslinking reaction. Single cell microgels were cultured in trans-well plates and allowed to recover from encapsulation for 24 hours following culture in chondrogenic differentiation medium with bi-weekly refreshments (Rodriguez Ruiz, Dicks et al. 2021). After seven days of differentiation, a portion of the soft single cell microgels were stiffened through controlled exposure of the single cell microgels to the crosslinking enzyme and hydrogen peroxide (in a process known as post-cure) (Kamperman, Henke et al. 2021). Viability of the single cell microgels was monitored using CAM and PI staining, chondrogenesis was analyzed with RT-qPCR, and immunofluorescent staining (anabolic cartilage marker COL2A1; hypertrophy and bone mineralization markers COL10 and SPP1) before and after 21 days of chondrogenesis. Stiffness of the single cell microgels was determined through nanoindentation.Results: Single cell microgels remained stable and supported hiMSC survival (>80%) throughout the 21 day chondrogenesis. While remaining non-proliferative, soft single cell microgels showed an increase in size of 1x (25µm) to 4x larger than initial encapsulation diameter which is not seen in stiff or post-cure microgels. Importantly, chondrogenic marker COL2 was expressed in >80% of cells (Fig. 1A) at day 21 in soft and post-cure microgels (Fig. 1B), while observed in only 35% of stiff microgels (Fig.1 C)(quantified expression is compared in Fig. 1G). Bone marker SPP1 was expressed in 60% of soft microgels (Fig. 1D) but not observed in any post-cure or stiff microgels (Fig. 1E-F)(quantified expression is compared in Fig. 1H). Hypertrophy marker COL10 was found in 85% of soft microgels (Fig. 1 D), 57% of post-cure microgels (Fig. 1F), and 20% of stiff microgels (Fig. 1E)(quantified expression is compared in Fig. 1I).Conclusions: We display that hiMSCs differentiated in single cell post-cure microgels show improved expression of cartilage extracellular matrix proteins. Post-cure microgel embedded chondrocyte-like cells display early commitment to COL2 expression as observed in soft microgels, while preventing SPP1 expression and reducing COL10 expression similar to stiff microgels. Therefore, we posit that time-dependent control of hiMSC microenvironment during chondrogenic differentiation in single cell microgels is an opportunity for the improved generation of chondrocyte-like cells and the analysis of cell fate decisions. In ongoing analysis of multi-model single cell sequencing we will identify the key factors that drive such differences and can improve neocartilage deposited by hiMSCs
RNA sequencing data integration reveals an miRNA interactome of osteoarthritis cartilage
No full textObjective To uncover the microRNA (miRNA) interactome of the osteoarthritis (OA) pathophysiological process in the cartilage.Methods We performed RNA sequencing in 130 samples (n=35 and n=30 pairs for messenger RNA (mRNA) and miRNA, respectively) on macroscopically preserved and lesioned OA cartilage from the same patient and performed differential expression (DE) analysis of miRNA and mRNAs. To build an OA-specific miRNA interactome, a prioritisation scheme was applied based on inverse Pearson’s correlations and inverse DE of miRNAs and mRNAs. Subsequently, these were filtered by those present in predicted (TargetScan/microT-CDS) and/or experimentally validated (miRTarBase/TarBase) public databases. Pathway enrichment analysis was applied to elucidate OA-related pathways likely mediated by miRNA regulatory mechanisms.Results We found 142 miRNAs and 2387 mRNAs to be differentially expressed between lesioned and preserved OA articular cartilage. After applying prioritisation towards likely miRNA-mRNA targets, a regulatory network of 62 miRNAs targeting 238 mRNAs was created. Subsequent pathway enrichment analysis of these mRNAs (or genes) elucidated that genes within the ‘nervous system development’ are likely mediated by miRNA regulatory mechanisms (familywise error=8.4×10−5). Herein NTF3 encodes neurotrophin-3, which controls survival and differentiation of neurons and which is closely related to the nerve growth factor.Conclusions By an integrated approach of miRNA and mRNA sequencing data of OA cartilage, an OA miRNA interactome and related pathways were elucidated. Our functional data demonstrated interacting levels at which miRNA affects expression of genes in the cartilage and exemplified the complexity of functionally validating a network of genes that may be targeted by multiple miRNAs.Pattern Recognition and Bioinformatic
A robust and standardized method to isolate and expand mesenchymal stromal cells from human umbilical cord
No full textBackground aims: Human umbilical cord–derived mesenchymal stromal cells (hUC-MSCs) are increasingly used in research and therapy. To obtain hUC-MSCs, a diversity of isolation and expansion methods are applied. Here, we report on a robust and standardized method for hUC-MSC isolation and expansion. Methods: Using 90 hUC donors, we compared and optimized critical variables during each phase of the multi-step procedure involving UC collection, processing, MSC isolation, expansion and characterization. Furthermore, we assessed the effect of donor-to-donor variability regarding UC morphology and donor attributes on hUC-MSC characteristics. Results: We demonstrated robustness of our method across 90 UC donors at each step of the procedure. With our method, UCs can be collected up to 6 h after birth, and UC-processing can be initiated up to 48 h after collection without impacting on hUC-MSC characteristics. The removal of blood vessels before explant cultures improved hUC-MSC purity. Expansion in Minimum essential medium α supplemented with human platelet lysate increased reproducibility of the expansion rate and MSC characteristics as compared with Dulbecco's Modified Eagle's Medium supplemented with fetal bovine serum. The isolated hUC-MSCs showed a purity of ∼98.9%, a viability of >97% and a high proliferative capacity. Trilineage differentiation capacity of hUC-MSCs was reduced as compared with bone marrow-derived MSCs. Functional assays indicated that the hUC-MSCs were able to inhibit T-cell proliferation demonstrating their immune-modulatory capacity. Conclusions: We present a robust and standardized method to isolate and expand hUC-MSCs, minimizing technical variability and thereby lay a foundation to advance reliability and comparability of results obtained from different donors and different studies.</p
Isolation and tracing of matrix-producing notochordal and chondrocyte cells using ACAN-2A-mScarlet reporter human iPSC lines
No full textInternational audienceThe development of human induced pluripotent stem cell (iPSC)–based regenerative therapies is challenged by the lack of specific cell markers to isolate differentiated cell types and improve differentiation protocols. This issue is particularly critical for notochordal-like cells and chondrocytes, which are crucial in treating back pain and osteoarthritis, respectively. Both cell types produce abundant proteoglycan aggrecan (ACAN), crucial for the extracellular matrix. We generated two human iPSC lines containing an ACAN-2A-mScarlet reporter. The reporter cell lines were validated using CRISPR-mediated transactivation and functionally validated during notochord and cartilage differentiation. The ability to isolate differentiated cell populations producing ACAN enables their enrichment even in the absence of specific cell markers and allows for comprehensive studies and protocol refinement. ACAN’s prevalence in various tissues (e.g., cardiac and cerebral) underscores the reporter’s versatility as a valuable tool for tracking matrix protein production in diverse cell types, benefiting developmental biology, matrix pathophysiology, and regenerative medicine
