Adipose-derived stem cells (ADSCs) exhibit promising potential for the treatment of various diseases, including osteoarthritis. Spheroids derived from ADSCs are a viable treatment option with enhanced anti-inflammatory effects and tissue repair capabilities.

SphereRing® is a rotating donut-shaped tube that efficiently produces large quantities of spheroids. However, accurately measuring spheroid size for spheroid quality assessment is challenging. This study aimed to develop an automated method for measuring spheroid size using deep learning through the ChatGPT Data Analyst for image recognition and processing.

The area, perimeter, and circularity of spheroids generated with the SphereRing system were analyzed using ChatGPT Data Analyst and ImageJ. Measurement accuracy was validated using Bland-Altman analysis and scatter plot correlation coefficients.

ChatGPT Data Analyst was consistent with ImageJ for all parameters. Bland-Altman plots demonstrated strong agreement; most data points were within the 95% limits.

The ChatGPT Data Analyst provides a reliable and efficient alternative for assessing spheroid quality. This method reduces human error and improves reproducibility to enhance spheroid quality control. Thus, this method has potential applications in regenerative medicine.

Human mesenchymal stromal cells (MSCs) possess regenerative potential due to pluripotency and paracrine functions. However, their stemness and immunomodulatory capabilities are sub-optimal in conventional two-dimensional (2D) culture.

To enhance the efficiency and therapeutic efficacy of MSCs, an in vivo-like 3D culture condition was applied.

MSCs were cultured on polystyrene (2D) or in a gellan gum-based 3D system. In vitro, prostaglandin-endoperoxide synthase 2, indoleamine-2,3-dioxygenase, heme oxygenase 1, and prostaglandin E synthase gene expression was quantified by quantitative real-time polymerase chain reaction. MSCs were incubated with lipopolysaccharide (LPS)-treated mouse splenocytes, and prostaglandin E2 and tumor necrosis factor-alpha levels were measured by enzyme linked immunosorbent assay. In vivo, LPS was injected into the lateral ventricle of mouse brain, and MSCs were administered intravenously the next day. Animals were sacrificed and analyzed on days 2 and 6.

Gellan gum polymer-based 3D culture significantly increased expression of octamer-binding transcription factor 4 and Nanog homeobox stemness markers in human MSCs compared to 2D culture. This 3D environment also heightened expression of cyclooxygenase-2 and heme-oxygenase 1, enzymes known for immunomodulatory functions, including production of prostaglandins and heme degradation, respectively. MSCs in 3D culture secreted more prostaglandin E2 and effectively suppressed tumor necrosis factor-alpha release from LPS-stimulated splenocytes and surpassed the efficiency of MSCs cultured in 2D. In a murine neuroinflammation model, intravenous injection of 3D-cultured MSCs significantly reduced ionized calcium-binding adaptor molecule 1 and glial fibrillary acidic protein expression, mitigating chronic inflammation more effectively than 2D-cultured MSCs.

The microenvironment established in 3D culture serves as an in vivo mimetic, enhancing the immunomodulatory function of MSCs. This suggests that engineered MSCs hold significant promise a potent tool for cell therapy.

The immune-modulatory effects of mesenchymal stromal cells (MSCs) are widely used to treat inflammatory disorders, with indoleamine 2,4-dioxygenase-1 (IDO-1) playing a pivotal role in suppressing stimulated T-cell proliferation. Taking that three-dimensional (3D) cultures enhance MSCs' anti-inflammatory properties compared with two-dimensional (2D) cultures, the differentially expressed miRNAs were examined. Thus, we identified hsa-miR-4662a-5p (miR-4662a) as a key inducer of IDO-1 via its suppression of bridging integrator-1 (BIN-1), a negative regulator of the IDO-1 gene. The IDO-1-inducing potential of miR-4662a was conserved across primary MSCs from various donors and sources but exhibited variability. Notably, iPSC-derived MSCs (iMSCs) demonstrated superior IDO-1 induction and immune-modulatory efficacy compared with their donor-matched primary MSCs. Accordingly, iMSCs expressing miR-4662a (4662a/iMSC) exhibited stronger suppressive effects on T-cell proliferation and more potent suppressive effects on graft-versus-host disease (GVHD), improving survival rates and reducing tissue damage in the liver and gut. Our results point to the therapeutic potential of standardized, off-the-shelf 4662a/iMSC as a robust immune-modulating cell therapy for GVHD.

Hair follicle cells reside within a complex extracellular matrix (ECM) environment in vivo, where physical and chemical cues regulate their behavior. The ECM is crucial for hair follicle development and regeneration, particularly through epithelial-mesenchymal interactions. Current in vitro models often fail to replicate this complexity, leading to inconsistencies in evaluating hair loss treatments. Advanced 3-dimensional (3D) culture systems that better mimic in vivo ECM dynamics are needed for more effective therapeutic assessments. Here, we introduce a 3D co-culture system designed to replicate in vivo ECM dynamics. The system incorporates primary dermal papilla cells from human patients, co-cultured with neonatal keratinocytes. This platform facilitates uniform spheroid formation through cell sliding and aggregation, enabling the evaluation of approximately 60 spheroids per well. The model is optimized for high-throughput screening, allowing precise assessments of hair-loss-inducing compounds under consistent conditions. We successfully generated dermal papilla cell and keratinocyte spheroids that closely resemble the native ECM structure, providing an optimal microenvironment for studying hair follicle biology. The 3D co-culture model supported efficient spheroid formation with consistent cellular organization and polarization, along with enhanced ECM-related gene expression crucial for hair follicle regeneration. Uniform spheroid formation and reproducibility were demonstrated across experiments. Overall, the novel 3D co-culture system provides a robust platform for replicating in vivo-like ECM conditions, enabling effective assessment of potential hair loss treatments through epithelial-mesenchymal interactions. Its high-throughput capacity, combined with reproducibility and ease of use, makes it a valuable tool for screening therapeutic candidates and advancing hair loss treatment development.

Mesenchymal stem cells (MSCs) play an important role in regenerative medicine and drug discovery due to their multipotential differentiation capabilities and immunomodulatory effects. Compared with traditional 2D cultures of MSCs, 3D cultures of MSCs have emerged as an effective approach to enhance cell viability, proliferation, and functionality, and provide a more relevant physiological environment. Here, we review the therapeutic potential of 3D-cultured MSCs, highlighting their roles in tissue regeneration and repair and drug screening. We further summarize successful cases that apply 3D MSCs in modeling disease states, enabling the identification of novel therapeutic strategies. Despite these promising applications, we discuss challenges that remain in the clinical translation of 3D MSC technologies, including stability, cell heterogeneity, and regulatory issues. We conclude by addressing these obstacles and emphasizing the need for further research to fully exploit the potential of 3D MSCs in clinical practice.

Neurodegenerative diseases (NDDs) present significant challenges due to limited treatment options, ethical concerns surrounding traditional animal models, and the time-consuming and costly process of using human-induced pluripotent stem cells (iPSCs). We addressed these issues by developing a 3D culture protocol for differentiating SH-SY5Y cells into glutamatergic neurons, enhancing physiological relevance with a 3D microarray culture plate. Our protocol optimized serum concentration and incorporated retinoic acid (RA) to improve differentiation. We analyzed the proportions of N-type and S-type cells, observing that RA in the maturation stage not only reduced cell proliferation but also enhanced the expression of MAP2 and VGLUT1, indicating effective neuronal differentiation. Our approach demonstrates the strong expression of glutamatergic neuron phenotypes in 3D SH-SY5Y neural spheroids, offering a promising tool for high-throughput NDD modeling and advancing drug discovery and therapeutic development. This method overcomes limitations associated with conventional 2D cultures and animal models, providing a more effective platform for NDD research.

Understanding the impact of various culturing strategies on the secretome composition of adipose-derived stromal cells (ASC) enhances their therapeutic potential. This study investigated changes in the secretome of perirenal ASC (prASC) under different conditions: normoxia, cytokine exposure, high glucose, hypoxia, and hypoxia with high glucose. Using mass spectrometry and enrichment clustering analysis, we found that normoxia enriched pathways related to extracellular matrix (ECM) organization, platelet degranulation, and insulin-like growth factor (IGF) transport and uptake. Cytokine exposure influenced metabolism, vascular development, and protein processing pathways. High glucose affected the immune system, metabolic processes, and IGF transport and uptake. Hypoxia impacted immune and metabolic processes and protein processing. Combined hypoxia and high glucose influenced the immune system, IGF transport and uptake, and ECM organization. Our findings highlight the potential of manipulating culturing conditions to produce secretomes with distinct protein and functional profiles, tailoring therapeutic strategies accordingly.

The therapeutic effects of extracellular vesicles (EVs) have been identified as a significant factor in intercellular communication in different disease treatments, including osteoarthritis (OA). Compared to the conventional approaches in treating OA, EV therapy is a non-invasive and cell-free method. However, improving the yield of EVs and their therapeutic effects are the main challenges for clinical applications. In this regard, researchers are using the EV engineering potential to overcome these challenges. New findings suggest that the co-culture strategy as an indirect EV engineering method efficiently increases EV production and quality. The co-culture of mesenchymal stem cells (MSCs) and chondrocytes has improved their chondrogenesis, anti-inflammatory effects, and regenerative properties which are mediated by EVs. Hence, co-culture engineering by considerable systems could be useful in producing engineered EVs for different therapeutic applications. Here, we review various co-culture approaches, including diverse direct and indirect, 2D and 3D cell cultures, as well as static and dynamic systems. Meanwhile, we suggest and discuss the advantages of combined strategies to achieve engineered EVs for OA treatment.

Human adipose tissue-derived stem/stromal cells (hASCs) are adult multipotent mesenchymal stem/stromal cells with immunomodulatory capacities. Here, we present up-to-date knowledge on the impact of different experimental and donor-related factors on hASC immunoregulatory functions in vitro. The experimental determinants include the immunological status of hASCs relative to target immune cells, contact vs. contactless interaction, and oxygen tension. Factors such as the ratio of hASCs to immune cells, the cellular context, the immune cell activation status, and coculture duration are also discussed. Conditioning of hASCs with different approaches before interaction with immune cells, hASC culture in xenogenic or xenofree culture medium, hASC culture in two-dimension vs. three-dimension with biomaterials, and the hASC passage number are among the experimental parameters that greatly may impact the hASC immunosuppressive potential in vitro, thus, they are also considered. Moreover, the influence of donor-related characteristics such as age, sex, and health status on hASC immunomodulation in vitro is reviewed. By analysis of the literature studies, most of the indicated determinants have been investigated in broad non-standardized ranges, so the results are not univocal. Clear conclusions cannot be drawn for the fine-tuned scenarios of many important factors to set a standard hASC immunopotency assay. Such variability needs to be carefully considered in further standardized research. Importantly, field experts' opinions may help to make it clearer.

The therapeutic application of multipotent mesenchymal stem cells (MSCs) encounters significant challenges, primarily stemming from their inadequate growth and limited self-renewal capabilities. Additionally, as MSCs are propagated, their ability to self-renew declines, and the exact cellular and molecular changes responsible for this are poorly understood. This study aims to uncover the complex molecular mechanisms that govern the self-renewal of primitive (p) MSCs.

We grew pMSCs using two types of medium, fetal bovine serum (FM) and xeno-free (XM), at both low passage (LP, P3) and high passage (HP, P20). To evaluate LP and HP pMSCs, we examined their physical characteristics, cell surface markers, growth rate, colony-forming ability, BrdU assays for proliferation, telomerase activity, and potential to differentiate into three lineages. Moreover, we conducted RNA-seq to analyze their transcriptome and MNase-seq analysis to investigate nucleosome occupancies.

When grown in FM, pMSCs underwent changes in their cellular morphology, becoming larger and elongated. This was accompanied by a decrease in the expression of CD90 and CD49f, as well as a reduction in CFE, proliferation rate, and telomerase activity. In addition, these cells showed an increased tendency to differentiate into the adipogenic lineage. However, when grown in XM, pMSCs maintained their self-renewal capacity and ability to differentiate into multiple lineages while preserving their fibroblastoid morphology. Transcriptomic analysis showed an upregulation of genes associated with self-renewal, cell cycle regulation, and DNA replication in XM-cultured pMSCs, while senescence-related genes were upregulated in FM-cultured cells. Further analysis demonstrated differential nucleosomal occupancies in self-renewal and senescence-related genes for pMSCs grown in XM and FM, respectively. These findings were confirmed by qRT-PCR analysis, which revealed alterations in the expression of genes related to self-renewal, cell cycle regulation, DNA replication, differentiation, and senescence. To understand the underlying mechanisms, we investigated the involvement of Wnt and TGFβ signaling pathways by modulating them with agonists and antagonists. This experimental manipulation led to the upregulation and downregulation of self-renewal genes in pMSCs, providing further insights into the signaling pathways governing the self-renewal and senescence of pMSCs.

Our study shows that the self-renewal potential of pMSCs is associated with the Wnt pathway, while senescence is linked to TGFβ.

Mesenchymal stromal cells (MSCs) have attracted scientific and medical interest due to their self-renewing properties, pluripotency, and paracrine function. However, one of the main limitations to the clinical application of MSCs is their loss of efficacy after transplantation in vivo. Various bioengineering technologies to provide stem cell niche-like conditions have the potential to overcome this limitation. Here, focusing on the stem cell niche microenvironment, studies to maximize the immunomodulatory potential of MSCs by controlling biomechanical stimuli, including shear stress, hydrostatic pressure, stretch, and biophysical cues, such as extracellular matrix mimetic substrates, are discussed. The application of biomechanical forces or biophysical cues to the stem cell microenvironment will be beneficial for enhancing the immunomodulatory function of MSCs during cultivation and overcoming the current limitations of MSC therapy.

Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) gain attention as a potent cell source in regenerative medicine and drug discovery. With the necessity of the demands for experimental models to create a more physiologically relevant model of the heart in vitro we herein investigate a 3D culturing platform and a method for assessing rhythm in hiPSC-CMs.

The 3D cell culture PAMCELL™ plate is designed to enable cells to attach exclusively to adhesive patterned areas. These cell adhesive zones, named as micro-patterned pads, feature micron silica beads that are surface-modified with the well-known arginyl-glycyl-aspartic acid (RGD) peptide. RGD binding to the surface of hiPSC-CMs facilitates cell-cell attachment and the formation of uniform-size spheroids, which is controlled by the diameter of the micro-patterned pads. The assessment and evaluation of 3D hiPSC-CMs beating pattern are carried out using reflective properties of retroreflective Janus micro-particle (RJP). These RJPs are modified with an antibody targeting the gap junction protein found on the surface of hiPSC-CM spheroids. The signal assessment system comprises a camera attached to an optical microscope and a white light source.

The 3D PAMCELL™ R100 culture plate efficiently generate approximately 350 uniform-sized hiPSC-CM spheroids in each well of a 96-well plate and supported a 20-day culture. Analysis of genes and protein expression levels reveal that iPSC-CM spheroids grown on PAMCELL™ R100 retain cardiac stem cell characteristics and functions, outperforming traditional 2D culture platform. Additionally, the RJPs enable monitoring and evaluation of in vitro beating properties of cardiomyocytes without using complex monitoring setup. The system demonstrates its capability to identify alteration in the rhythmic activity of cardiac cells when exposed to ion channel blockers, nifedipine and E4031.

The integration of the 3D culture method and RJPs in this study establishes a platform for evaluating the rhythmic properties of 3D hiPSC-CMs. This approach holds significant potential for identifying arrhythmias or other cardiac abnormalities, ultimately contributing to the development of more effective therapies for heart diseases.

Hepatic fibrosis/cirrhosis is a significant health burden worldwide, resulting in liver failure or hepatocellular carcinoma (HCC) and accounting for many deaths each year. The pathogenesis of hepatic fibrosis/cirrhosis is very complex, which makes treatment challenging. Endogenous mesenchymal stromal cells (MSCs) have been shown to play pivotal roles in the pathogenesis of hepatic fibrosis. Paradoxically, exogenous MSCs have also been used in clinical trials for liver cirrhosis, and their effectiveness has been observed in most completed clinical trials. There are still many issues to be resolved to promote the use of MSCs in the clinic in the future. In this review, we will examine the controversial role of MSCs in the pathogenesis and treatment of hepatic fibrosis/cirrhosis. We also investigated the clinical trials involving MSCs in liver cirrhosis, summarized the parameters that need to be standardized, and discussed how to promote the use of MSCs from a clinical perspective.

Perianal fistulas, characterised as granulomatous inflammation of fistulas around the anal canal, are associated with significant morbidity resulting in a negative impact on quality of life and a tremendous burden to the healthcare system. Treatment of anal fistulas usually consists of anal surgery; however, results of closure rates are not satisfactory especially with complex perianal fistulas, after which many patients may suffer from anal incontinence. Recently, the administration of mesenchymal stem cells (MSCs) has shown promising efficacy. Herein, we aim to explore whether MSCs are effective for complex perianal fistulas and if they have either short-term, medium-term, long-term or over-long-term efficacy. Additionally, we want to elucidate whether factors such as drug dosage, MSC source, cell type, and disease aetiology influence treatment efficacy. We searched four online databases and analysed data based on information within the clinical trials registry. The outcomes of eligible trials were analysed with Review Manager 5.4.1. Relative risk and related 95% confidence interval were calculated to compare the effect between the MSCs and control groups. In addition, the Cochrane risk of bias tool was applied to evaluate the bias risk of eligible studies. Meta-analyses showed that therapy with MSCs was superior to conventional treatment for complex perianal fistulas in short-, long- and over-long-term follow-up phases. However, there was no statistical difference in treatment efficacy in the medium term between the two methods. Subgroup meta-analyses showed factors including cell type, cell source and cell dosage were superior compared to the control, but there was no significant difference between different experimental groups of those factors. Besides, local MSCs therapy has shown more promising results for fistulas as a result of Crohn's Disease (CD). Although we tend to maintain that MSCs therapy is effective for cryptoglandular fistulas equally, more studies are needed to confirm this conclusion in the future.

MSCs Transplantation could be a new therapeutic method for complex perianal fistulas of both cryptoglandular and CD origin showing high efficacy in the short-term to over-long-term phases, as well as high efficacy in sustained healing. The difference in cell types, cell sources and cell dosages did not influence MSCs' efficacy.

Therapeutic use of multipotent mesenchymal stem cells (MSCs) is hampered due to poor growth and limited self-renewal potential. The self-renewal potential of MSCs is also affected during propagation and changes are poorly understood. This study investigated the molecular mechanism involved in the self-renewal of primitive (p) MSCs.

pMSCs were cultured to low passage (LP), P3, and high passage (HP), P20, in fetal bovine serum medium (FM) and xeno-free medium (XM). The characteristics of LP and HP pMSCs were evaluated for morphology, expression of cell surface markers, doubling time (DT), colony forming efficiency (CFE), proliferation by BrdU assay, telomerase activity and trilineage differentiation. We then examined transcriptome and nucleosome occupancies using RNA-seq and MNase-seq, respectively analyses.

pMSCs grown in FM gradually changed morphology to large elongated cells and showed a significant reduction in the expression of CD90 and CD49f, CFE, proliferation, and telomerase activity. In addition, cells had a greater propensity to differentiate into the adipogenic lineage. In contrast, pMSCs grown in XM maintained small fibroblastoid morphology, self-renewal, and differentiation potential. Transcriptomic analysis showed upregulation of genes involved in self-renewal, cell cycle, and DNA replication in XM-grown pMSCs. Whereas senescence genes were upregulated in cells in FM. MNase-seq analysis revealed less nucleosomal occupancies in self-renewal genes and senescence genes in pMSCs grown in XM and FM, respectively. The expression of selected genes associated with self-renewal, cell cycle, DNA replication, differentiation, and senescence was confirmed by qRT-PCR. These results led us to propose signaling pathways involved in the self-renewal and senescence of pMSCs.

We conclude that the self-renewal potential of pMSCs is controlled by WNT and VEGF/PDGF, but TGFβ and PI3K signaling induce senescence.

It is unclear whether extracellular vesicles (EVs) from mesenchymal stem cells (MSCs) have a direct protective effect on pancreatic islets. In addition, whether culturing MSCs in three dimensions (3D) instead of a monolayer (2D) can induce changes in the cargo of EVs that facilitate the polarization of macrophages into an M2 phenotype has not been investigated. We sought to determine whether EVs from MSCs cultured in 3D can prevent inflammation and dedifferentiation in pancreatic islets and, if so, whether the protective effect is superior to that of EVs from 2D MSCs. Human umbilical cord blood- (hUCB-) MSCs cultured in 3D were optimized according to cell density, exposure to hypoxia, and cytokine treatment based on the ability of the hUCB-MSC-derived EVs to induce the M2 polarization of macrophages. Islets isolated from human islet amyloid polypeptide (hIAPP) heterozygote transgenic mice were cultured in serum-deprived conditions with hUCB-MSC-derived EVs. EVs derived from 3D hUCB-MSCs had more abundant microRNAs involved in M2 polarization of macrophages and had an enhanced M2 polarization ability on macrophages, which was optimized when the 3D culture condition was 2.5 × 10⁴ cells per spheroid without preconditioning with hypoxia and cytokine exposure. When islets isolated from hIAPP heterozygote transgenic mice were cultured in serum-deprived conditions with hUCB-MSC-derived EVs, the EVs derived from 3D hUCB-MSCs suppressed the expression of proinflammatory cytokines and caspase-1 in pancreatic islets and increased the proportion of M2-polarized islet-resident macrophages. They improved glucose-stimulated insulin secretion, reduced the expression of Oct4 and NGN3, and induced the expression of Pdx1 and FoxO1. The greater suppression of IL-1β, NLRP3 inflammasome, caspase-1, and Oct4 and induction of Pdx1 and FoxO1 were found in islets cultured with the EVs derived from 3D hUCB-MSCs. In conclusion, EVs derived from 3D hUCB-MSCs optimized for M2 polarization attenuated nonspecific inflammation and preserved β-cell identity of pancreatic islets.

Extracellular vesicles (EVs) have therapeutic effects on osteoarthritis (OA). Some recent strategies could elevate EV's therapeutic properties including cell aggregation, co-culture, and 3D culture. It seems that a combination of these strategies could augment EV production and therapeutic potential. The current study aims to evaluate the quantity of EV yield and the therapeutic effect of EVs harvested from rabbit mesenchymal stem cells (MSCs) aggregates, chondrocyte aggregates, and their co-aggregates in a dynamic 3D culture in a rat osteoarthritis model. MSC and chondrocytes were aggregated and co-aggregated by spinner flasks, and their conditioned medium was collected. EVs were isolated by size exclusion chromatography and characterized in terms of size, morphology and surface markers. The chondrogenic potential of the MSC-ag, Cho-ag and Co-ag EVs on MSC micromass differentiation in chondrogenic media were assessed by qRT-PCR, histological and immunohistochemical analysis. 50 μg of MSC-ag-EVs, Cho-ag-EVs and Co-ag-EVs was injected intra-articularly per knee of OA models established by monoiodoacetate in rats. After 8 weeks follow up, the knee joints were harvested and analyzed by radiographic, histological and immunohistochemical features. MSC/chondrocyte co-aggregation in comparison to MSC or chondrocyte aggregation could increase EV yield during dynamic 3D culture by spinner flasks. Although MSC-ag-, Cho-ag- and Co-ag-derived EVs could induce chondrogenesis similar to transforming growth factor-beta during in vitro study, Co-ag-EV could more effectively prevent OA progression than MSC-ag- and Cho-ag-EVs. Our study demonstrated that EVs harvested from the co-aggregation of MSCs and chondrocytes could be considered as a new therapeutic potential for OA treatment.

Liver fibrosis is a common result of most chronic liver diseases, and advanced fibrosis often leads to cirrhosis. Currently, there is no effective treatment for liver cirrhosis except liver transplantation. Therefore, it is important to carry out antifibrosis treatment to reverse liver damage in the early stage of liver fibrosis. Mesenchymal stem cells (MSCs) are the most widely used stem cells in the field of regenerative medicine. The preclinical and clinical research results of MSCs in the treatment of liver fibrosis and cirrhosis show that MSC administration is a promising treatment for liver fibrosis and cirrhosis. MSCs reverse liver fibrosis and increase liver function mainly through differentiation into hepatocytes, immune regulation, secretion of cytokines and other nutritional factors, reduction of hepatocyte apoptosis, and promotion of hepatocyte regeneration. Recently, many studies provided a variety of new methods and strategies to improve the effect of MSCs in the treatment of liver fibrosis. In this review, we summarized the current effective methods and strategies and their potential mechanisms of MSCs in the treatment of liver fibrosis, as well as the current research progress in clinical practice. We expect to achieve complete reversal of liver injury with MSC-based therapy in the future.