Figure - available from: Stem Cell Research & Therapy
This content is subject to copyright. Terms and conditions apply.
Initial cell number modulates the expression of different mechanosensing proteins. a Western blot analysis revealed that initial cell number significantly impacts the expression of Cav-1, N-cad, β-cat, and FN. b Quantification of western blot analysis data. Values are normalized to GAPDH. Statistical analysis is based on a one-way ANOVA tool of OriginPro. Error bars are standard error of the mean, and different aggregates in one group are compared to 70 k in the same group (*p < 0.05, **p < 0.01, ***p < 0.001). c Immunofluorescence staining for Cav-1 and N-cad in sections of pellets after 7 days of differentiation
Source publication
Background
Although mesenchymal stem/stromal cell (MSC) chondrogenic differentiation has been thoroughly investigated, the rudiments for enhancing chondrogenesis have remained largely dependent on external cues. Focus to date has been on extrinsic variables such as soluble signals, culture conditions (bioreactors), and mechanical stimulation. Howev...
Citations
... Importantly, the meso-scale size of the generated spheroids (i.e. diameter 500-1000 μm using 50,000 cells/spheroid) allowed to minimize the formation of core tissue necrosis, (i.e. a problem that typically occurs during the culture of macro-scale tissues > 1 mm diameter, normally generated with >100,000 BMSC) [61,62] and at the same time to perform assessments requiring sufficient tissue quantity (i.e., mass spectrometry or mechanical measurements) [63]. ...
... Spheroid size emerges as a significant factor influencing nutrient and oxygen supply, as well as mechanical stresses arising from cell-to-cell interactions. This, in turn, modifies gene expression and underscores the importance of carefully controlling the 3D culture environment for optimal outcomes, as well as choosing the most suitable spheroid generation technique ( Figure 2) [8]. The utilization of 3D MSC models has provided valuable insights into the fundamental characteristics of MSCs and has paved the way for the development of diverse treatment approaches. ...
To more accurately replicate the in vivo three-dimensional (3D) mesenchymal stem cell (MSC) niche and enhance cellular phenotypes for superior in vivo treatments, MSC functionalization through in vitro 3D culture approaches has gained attention. The organization of MSCs in 3D spheroids results in altered cell shape, cytoskeleton rearrangement, and polarization. Investigations have revealed that the survival and secretory capability of MSCs are positively impacted by moderate hypoxia within the inner zones of MSC spheroids. The spheroid hypoxic microenvironment enhances the production of angiogenic and anti-apoptotic molecules, including HGF, VEGF, and FGF-2. Furthermore, it upregulates the expression of hypoxia-adaptive molecules such as CXCL12 and HIF-1, inhibiting MSC death. The current review focuses on the latest developments in fundamental and translational research concerning three-dimensional MSC systems. This emphasis extends to the primary benefits and potential applications of MSC spheroids, particularly in the context of breast cancer and customized regenerative therapies.
... Furthermore, our single-cell data revealed high expression of FZD4 in the mesenchymal condensate, a finding supported by RNA-ISH (Fig. 5a,d,e). This supports the suggestion from in vitro studies that FZD4 has a role in initiating chondrogenesis when mesenchymal condensate reaches a critical mass 68 . ...
Human limbs emerge during the fourth post-conception week as mesenchymal buds, which develop into fully formed limbs over the subsequent months¹. This process is orchestrated by numerous temporally and spatially restricted gene expression programmes, making congenital alterations in phenotype common². Decades of work with model organisms have defined the fundamental mechanisms underlying vertebrate limb development, but an in-depth characterization of this process in humans has yet to be performed. Here we detail human embryonic limb development across space and time using single-cell and spatial transcriptomics. We demonstrate extensive diversification of cells from a few multipotent progenitors to myriad differentiated cell states, including several novel cell populations. We uncover two waves of human muscle development, each characterized by different cell states regulated by separate gene expression programmes, and identify musculin (MSC) as a key transcriptional repressor maintaining muscle stem cell identity. Through assembly of multiple anatomically continuous spatial transcriptomic samples using VisiumStitcher, we map cells across a sagittal section of a whole fetal hindlimb. We reveal a clear anatomical segregation between genes linked to brachydactyly and polysyndactyly, and uncover transcriptionally and spatially distinct populations of the mesenchyme in the autopod. Finally, we perform single-cell RNA sequencing on mouse embryonic limbs to facilitate cross-species developmental comparison, finding substantial homology between the two species.
... Multiparameter deformability cytometry classifies cells according to multiple biophysical characteristics, such as size, deformability, and morphology, through microfluidic inertial focusing, hydrodynamic stretching of cells, and high-speed video recording [140]. Since MSC differentiation results in a reorganisation of lamin A/C and an increase in heterochromatin levels [141], MSC stiffness is also directly correlated with the extent of chondrogenic differentiation [142]. The combination of small cell size, low cell stiffness, and high nuclear membrane fluctuations is indicative of high MSC clonogenicity and multipotency, and future cell isolation techniques may select for this subpopulation of MSCs [138]. ...
Articular cartilage defects commonly result from trauma and are associated with significant morbidity. Since cartilage is an avascular, aneural, and alymphatic tissue with a poor intrinsic healing ability, the regeneration of functional hyaline cartilage remains a difficult clinical problem. Mesenchymal stem cells (MSCs) are multipotent cells with multilineage differentiation potential, including the ability to differentiate into chondrocytes. Due to their availability and ease of ex vivo expansion, clinicians are increasingly applying MSCs in the treatment of cartilage lesions. However, despite encouraging pre-clinical and clinical data, inconsistencies in MSC proliferative and chondrogenic potential depending on donor, tissue source, cell subset, culture conditions, and handling techniques remain a key barrier to widespread clinical application of MSC therapy in cartilage regeneration. In this review, we highlight the strategies to manage the heterogeneity of MSCs ex vivo for more effective cartilage repair, including reducing the MSC culture expansion period, and selecting MSCs with higher chondrogenic potential through specific genetic markers, surface markers, and biophysical attributes. The accomplishment of a less heterogeneous population of culture-expanded MSCs may improve the scalability, reproducibility, and standardisation of MSC therapy for clinical application in cartilage regeneration.
... In the present study, the fused clusters exhibit liquid-like properties that lead to rounding with dynamics that are of the same order of magnitude as those of the rounding of individual clusters. The difference between the "arrested coalescence" seen in stem cell aggregates and our SMC cluster "liquid-like rounding" may be attributable to differences in internal structure 49 . ...
Smooth muscle cells (SMCs) are mural cells that play a vital contractile function in many tissues. Abnormalities in SMC organization are associated with many diseases including atherosclerosis, asthma, and uterine fibroids. Various studies have reported that SMCs cultured on flat surfaces can spontaneously form three-dimensional clusters whose organization resembles that encountered in some of these pathological settings. Remarkably, how these structures form remains unknown. Here we combine in vitro experiments and physical modeling to show that three-dimensional clusters initiate when cellular contractile forces induce a hole in a flat SMC sheet, a process that can be modeled as the brittle fracture of a viscoelastic material. The subsequent evolution of the nascent cluster can be modeled as an active dewetting process with cluster shape evolution driven by a balance between cluster surface tension, arising from both cell contractility and adhesion, and cluster viscous dissipation. The description of the physical mechanisms governing the spontaneous emergence of these intriguing three-dimensional clusters may offer insight into SMC-related disorders.
... Addition of cells in the hydrogels resulted in a decrease in stiffness in all sample conditions over time, similar to what has been reported in other types of hydrogels following cell encapsulation. 65 A plausible explanation for the G′ decrease would be that the presence of the softer than the hydrogel cells contribute to the overall stiffness of the samples. The minimum G′ value measured for the control hydrogels was close to 0.9 kPa, while the elastic modulus of single stem cells is around 0.5 kPa or lower. ...
Mesenchymal stem cells (MSCs) are the most prominent type of adult stem cells for clinical applications. Three-dimensional (3D) cultivation of MSCs in biomimetic hydrogels provides a more physiologically relevant cultivation microenvironment for in vitro testing and modeling, thus overcoming the limitations of traditional planar cultivation methods. Cellulose nanofibers are an excellent candidate biomaterial for synthesis of hydrogels for this application, due to their biocompatibility, tunable properties, availability, and low cost. Herein, we demonstrate the capacity of hydrogels prepared from 2,2,6,6-tetramethylpiperidine-1-oxyl -oxidized and subsequently individualized cellulose-nanofibrils to support physiologically relevant 3D in vitro cultivation of human MSCs at low solid contents (0.2-0.5 wt %). Our results show that MSCs can spread, proliferate, and migrate inside the cellulose hydrogels, while the metabolic activity and proliferative capacity of the cells as well as their morphological characteristics benefit more in the lower bulk cellulose concentration hydrogels.
... 3D spheroid cultures which involve spontaneous cell assemblage and condensation processes enable an advance spatial organization with improved cellular interactions, tropic secretions, and immunomodulatory abilities [14]. The role of cell condensation in promoting chondrogenesis and endochondral ossification is also well established [15,16]. Recent evidence suggests that the complex cellular architecture acquired in spheroid culture determines the osteocyte fate of MSC [17]. ...
Background:
Human mesenchymal stem cells are being used for various regenerative applications in past decades. This study chronicled a temporal profile of the transcriptional pattern and promoter methylation status of the osteogenic related gene in dental pulp stem cells (DPSCs) derived from 3-dimensional spheroid culture (3D) vis a vis 2-dimensional (2D) monolayer culture upon osteogenic induction.
Methods:
Biomimetic properties of osteogenesis were determined by alkaline phosphatase assay and alizarin red staining. Gene expression and promoter methylation status of osteogenic genes such as runt-related transcription factor-2, collagen1α1, osteocalcin (OCN), and DLX5 (distal-homeobox) were performed by qPCR assay and bisulfite sequencing, respectively. Furthermore, µ-Computed tomography (micro-CT) was performed to examine the new bone formation in critical-sized rat calvarial bone defect model.
Results:
Our results indicated a greater inclination of spheroid culture-derived DPSCs toward osteogenic lineage than the monolayer culture. The bisulfite sequencing of the promoter region of osteogenic genes revealed sustenance of low methylation levels in DPSCs during the progression of osteogenic differentiation. However, the significant difference in the methylation pattern between 2D and 3D derived DPSCs were identified only for OCN gene promoter. We observed differences in the mRNA expression pattern of epigenetic writers such as DNA methyltransferases (DNMTs) and methyl-cytosine dioxygenases (TET) between the two culture conditions. Further, the DPSC spheroids showed enhanced new bone formation ability in an animal model of bone defect compared to the cells cultivated in a 2D platform which further substantiated our in-vitro observations.
Conclusion:
The distinct cellular microenvironment induced changes in DNA methylation pattern and expression of epigenetic regulators such as DNMTs and TETs genes may lead to increase expression of osteogenic markers in 3D spheroid culture of DPSCs which make DPSCs spheroids suitable for osteogenic regeneration compared to monolayers.
... We hypothesised that the increased proliferation rate on 0.82 and 5.75 kPa hydrogels might be associated with cells aggregation which is known to improve cell viability and proliferation. 60 The effect of stiffness in directing MSCs behaviour was first demonstrated by Engler et al. 25 Their work suggested that, when pre-conditioned on a specific stiffness, MSCs couldn't be reprogrammed even with the use of growth factors. Park et al. highlighted the importance of stiffness, in particular of soft substrates (1 kPa), in directing MSCs towards the chondrogenic and adipogenic lineage; however, they warned this effect may not be specific for only one lineage, and biochemical factors such as TGF-β are required. ...
Mesenchymal stem cells (MSCs) hold great promise for the treatment of cartilage related injuries. However, selectively promoting stem cell differentiation in vivo is still challenging. Chondrogenic differentiation of MSCs usually requires the use of growth factors that lead to the overexpression of hypertrophic markers. In this study, for the first time the effect of stiffness on MSC differentiation has been tested without the use of growth factors. Three-dimensional collagen and alginate scaffolds were developed and characterised. Stiffness significantly affected gene expression and ECM deposition. While, all hydrogels supported chondrogenic differentiation and allowed deposition of collagen type II and aggrecan, the 5.75 kPa hydrogel showed limited production of collagen type I compared to the other two formulations. These findings demonstrated for the first time that stiffness can guide MSCs differentiation without the use of growth factors within a tissue engineering scaffold suitable for the treatment of cartilage defects.
... In turn, while a number of studies indicated BMP4 as a crucial factor in chondrogenic differentiation of ASCs, Shu et al. proved that its role is not as significant as that of BMP2, which seems to be supported by findings of our study [46][47][48]. Lower levels of ASS1 were associated with lower number of aggregated cells in chondrocyte differentiated MSCs, which finds confirmation in our study as the final chondrocyte spheroids were significantly smaller than the initial 3D cultured ASCs [49]. KRT7 upregulation in MSC cultures was associated with exposure to tumor-associated factors and progression towards carcinoma-associated fibroblast-like phenotype [50]. ...
The interest in stem cell research continuously increased over the last decades, becoming one of the most important trends in the 21st century medicine. Stem cell-based therapies have a potential to become a solution for a range of currently untreatable diseases, such as spinal cord injuries, type I diabetes, Parkinson’s disease, heart disease, stroke, and osteoarthritis. Hence, this study, based on canine material, aims to investigate the molecular basis of adipose-derived stem cell (ASC) differentiation into chondrocytes, to serve as a transcriptomic reference for further research aiming to introduce ASC into treatment of bone and cartilage related diseases, such as osteoarthritis in veterinary medicine. Adipose tissue samples were harvested from a canine specimen subjected to a routine ovariohysterecromy procedure at an associated veterinary clinic. The material was treated for ASC isolation and chondrogenic differentiation. RNA samples were isolated at day 1 of culture, day 30 of culture in unsupplemented culture media, and day 30 of culture in chondrogenic differentiation media. The resulting RNA was analyzed using RNAseq assays, with the results validated by RT-qPCR. Between differentiated chondrocytes, early and late cultures, most up- and down-regulated genes in each comparison were selected for further analysis., there are several genes (e.g., MMP12, MPEG1, CHI3L1, and CD36) that could be identified as new markers of chondrogenesis and the influence of long-term culture conditions on ASCs. The results of the study prove the usefulness of the in vitro culture model, providing further molecular insight into the processes associated with ASC culture and differentiation. Furthermore, the knowledge obtained could be used as a molecular reference for future in vivo and clinical studies.
... The hardness of the stacked bovine myoblast sheets increased with the extend in the culture days. Previous studies on cell spheroid have reported similar results 31,32 . To investigate the reason for this increase in hardness, HE and AZAN staining were performed. ...
In the production of cell-based meat, it is desirable to reduce animal-derived materials as much as possible to meet the challenges of sustainability. Here, we demonstrate the “cell sheet-based meat”: scaffold-free cell-based meat using cell sheet technology and characterize its texture and nutrients. Bovine myoblast cell sheets were prepared using temperature-responsive culture dishes (TRCDs) and 10 stacked cell sheets to fabricate three-dimensional tissue of 1.3–2.7 mm thickness. Hardness was increased by incubation on the TRCD and was further increased by boiling as is characteristic of natural meat. The wet weight percentage of total protein in the cell sheet was about half that of beef. In this method, large-sized items of cell sheet-based meat were also created by simply scaling up the TRCD. This method promises an environment-friendly food product.
