Stem Cells International最新文献

Background: At present, healthcare facilities often face blood shortages because of the low supply of donated blood relative to the high demand. Therefore, efforts to develop red blood cell (RBC) production methods have gained traction. In this work, Lin^-CD45^-CD133⁺ cells were isolated from human umbilical cord blood (UCB) and subsequently differentiated into erythrocytes in vitro in serum-free culture medium.

Methods: Lin^-CD45^-CD133⁺ cells were prepared from mononuclear cells (MNCs) using magnetic-activated cell sorting (MACS). The characteristics of Lin^-CD45^-CD133⁺ cells were confirmed using flow cytometry analysis, colony-forming unit (CFU) assays, morphological analysis, immunocytochemistry (ICC) analysis, and real-time fluorescent quantitative polymerase chain reaction (RT-PCR). Erythrocytes were differentiated in serum-free medium supplemented with stem cell factor (SCF), interleukin-3 (IL-3), erythropoietin (EPO), and FK506 for 13 days, after which autoplasma derived from UCB was added at a concentration of 5% beginning on day 14. Erythroid differentiation and maturation were examined using electron microscopy and flow cytometric analysis.

Results: Lin^-CD45^-CD133⁺ cells were successfully obtained from UCB. These cells were slightly smaller than normal RBCs and had a high nucleus-to-cytoplasm ratio. Oct-4 and Nanog were expressed at both the mRNA and protein levels in Lin^-CD45^-CD133⁺ cells. Most of the colonies were burst-forming unit-erythroid (BFU-E). After 7 days of in vitro culture, the Lin^-CD45^-CD133⁺ cells were negative for CD133 expression and positive for CD45 expression. The percentage of CD71⁺ cells gradually increased, peaked on day 10, and then started decreasing on day 13. The percentage of CD235a⁺ cells increased gradually after day 7 and peaked on day 13. CD240 expression was detected on day 18, with the highest level detected on day 20. The number of erythroid cells increased persistently during differentiation, and their morphology was consistent with that of normal erythrocytes.

Conclusion: An ex vivo culture system was developed that can generate human erythrocytes from Lin^-CD45^-CD133⁺ cells isolated from human UCB.

The development of robust and scalable culture systems is essential for the clinical-scale production of human umbilical cord (UC)-derived mesenchymal stem/stromal cells (MSCs) (UC-MSCs). While various basal and serum-free media are commercially available, systematic comparisons of their efficacy in supporting the expansion and functional properties of UC-MSCs remain limited. In this study, we conducted a comprehensive evaluation of multiple culture systems, including basal media (α-MEM, DMEM, and DMEM/F12) supplemented with human platelet lysate (HPL), and commercial serum-free media (Corning MSC Xeno-Free SFM, NutriStem XF Medium, Prime-XV MSC Expansion XSFM), for their ability to sustain UC-MSCs proliferation, maintain phenotypic properties, and support functional potency. The results demonstrated that all basal media supported cell growth, with α-MEM (Gibco) and DMEM/F12 showing superior performance over DMEM. Among serum-free formulations, Prime-XV with 2% HPL yielded the highest primary culture output and the shortest population doubling (PD) time (PDT) during passaging. Notably, cells expanded in commercial serum-free media exhibited reduced diameter and higher uniformity. Functional analyses revealed that NutriStem XF Medium supplemented with 2% HPL elicited the strongest immunomodulatory effects in mixed lymphocyte reactions (MLRs). Furthermore, all media maintained trilineage differentiation capacity and satisfied International Society for Cellular Therapy (ISCT) phenotypic criteria. Critically, no tumorigenic potential was detected in vitro or in vivo. Large-scale manufacturing using the selected medium (NutriStem XF + 2% HPL) confirmed consistent expansion kinetics, high viability, stable marker expression, and functional potency across seven production batches. This study provides a rigorous and clinically relevant framework for selecting culture media that ensure both scalability and functional integrity of UC-MSCs, highlighting the promise of serum-free systems for therapeutic manufacturing.

Background: Osteogenic differentiation is a crucial process in which bone marrow mesenchymal stem cells (BMSCs) differentiate into osteoblasts, involving the regulation of multiple genes and signaling pathways. The TSC22D3 gene plays an important role in various biological processes (BPs), but its specific function in osteogenic differentiation remains unclear. This study aims to explore the regulatory role of the TSC22D3 gene in osteogenic differentiation and its molecular mechanisms.

Methods: By analyzing microarray datasets (GSE12266, GSE18043, and GSE80614), the limma package was used to screen for differentially expressed genes (DEGs). Combined with Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses, key genes and signaling pathways related to osteogenic differentiation were identified. Further, through protein-protein interaction (PPI) network analysis and the Finding Regulatory Elements by Differential Expression and Network-Based Statistical Analysis (FRIEND) method, TSC22D3 was screened out as a core hub gene. For experimental validation, the bioinformatics analysis results were intersected with the transcriptome sequencing data from our research group to further confirm the core molecules. Lentivirus-mediated interference technology was used to downregulate and overexpress TSC22D3 expression, and the impact of TSC22D3 on osteogenic differentiation was assessed through RT-qPCR, Western blotting, alkaline phosphatase (ALP) staining, phalloidin staining, and calcium deposition assays.

Results: TSC22D3 is significantly upregulated during osteogenic differentiation; its downregulation can lead to reduced expression of osteogenic differentiation marker genes (such as runt-related transcription factor 2 [Runx2], osterix [OSX], osteocalcin [OCN], and osteopontin [OPN]), as well as a significant decrease in ALP activity and calcium deposition. GO and KEGG analyses indicate that TSC22D3 is closely associated with pathways including the cell cycle, cytoskeleton, and WNT signaling. Furthermore, Gene Set Enrichment Analysis (GSEA) analysis has further revealed the potential regulatory mechanism of TSC22D3 in osteogenic differentiation. Rescue experiments have confirmed that TSC22D3 can promote the osteogenic differentiation of BMSCs and induce the rearrangement of cytoskeletal structure.

Conclusion: This study reveals that TSC22D3 is essential for osteogenic differentiation. Its upregulation promotes osteogenic marker expression, ALP activity, and calcium deposition, while its downregulation inhibits these processes. TSC22D3 affects cytoskeletal rearrangement during osteogenesis.

Therapies utilizing human mesenchymal stromal cells (MSCs) are advancing through clinical trials, emphasizing the need for reliable, scalable, and cost-efficient manufacturing processes to support the lot sizes necessary for commercial-scale production. Wharton's jelly MSCs (WJMSCs) are valued for their regenerative abilities and immunomodulatory and anti-inflammatory properties, which contribute to tissue repair. With growing therapeutic demand, the production of WJMSCs must scale to yield billions of cells while maintaining their essential characteristics-identity, purity, and potency-necessary for clinical and regulatory compliance. Achieving such magnitude of expansion entails the utilization of current good manufacturing practice (cGMP)-compliant scalable culture systems that allow bioprocess control and monitoring. This study aimed to establish a scalable serum-/xeno-free expansion process representing a critical step towards a cGMP-compliant large-scale production platform for WJMSC-based clinical applications. Using our in-house GMP-manufactured WJMSCs, which were tested in a Phase Ib clinical trial (NCT03158896), we have previously optimized various culture parameters using a microcarrier (MC)-based three-dimensional (3D) culture system in spinner flasks and demonstrated successful WJMSC expansion. In the present study, we successfully translated culture conditions to a 2 L followed by a STR50 (50 L) stirred-tank bioreactor (BR) (STR), adhering to cGMP requirements. The culture system in the 2 and 50 LBRs supported cell concentrations of approximately 1.2 x 10⁶ cells/mL and attained 24-fold and 27-fold expansion, respectively, with a yield of approximately 37 billion cells in the 50 L culture system after 7 days with a 95% harvest efficiency. Following expansion, WJMSCs preserved their characteristic phenotypes, differentiation potential, chromosomal stability, functional capabilities, and sterility across all tested culture systems. We conclude that the large-scale expansion process of WJMSCs in the STR described herein is highly adaptable to the scale necessary to fulfill the commercial demand for high quality clinical-grade MSCs.

Cancer is a predominant testimony of human departure in a global way. Current therapeutic strategies are not sufficient, and thereby exploring a new approach with high efficacy and influence is desired. The intention of this research is to distinguish a novel therapeutic burgeon in colon cancer plus employing the human mesenchymal stem cells (hAMSCs) secretome as a new tool in colon cancer therapy. For this purpose, 30 pieces from patients afflicted with colon cancer were provided. The expressing level of IRSp53 was evaluated using quantitative real-time PCR (qRT-PCR). Then, a coculture procedure utilizing six well plates transwell was applied. Since 72 h, tumor increment was surveyed in HT-29 cells treated by hAMSCs through the EGFR/c-Src/IRSp53/p-AKT/p-Stat3/cyclin D1 signaling cascade. Our results indicated IRSp53 upregulation in patients suffering colon cancer and reduction of EGFR/c-Src/IRSp53/p-AKT/p-Stat3/cyclin D1 signaling pathway, which led to suppression of cell proliferation in the hAMSCs-treated HT-29 colon cancerous cells. We also found tumor growth suppression as well as IRSp53 expression in hAMSCs-treated HT-29 colon cancerous cell line using a 3D cell culturing technique. Our study's findings indicate that colon cancer therapy could benefit from targeting IRSp53 and that MSCs could be a valuable therapeutic option for stopping the proliferation of colon cancer cells. This could be achieved through the EGFR/c-Src/IRSp53/p-AKT/p-Stat3/cyclin D1 signaling pathway.

Diabetic kidney disease (DKD) is characterized by a continuous decline in renal function and progressive fibrosis, making it a leading cause of end-stage kidney disease with limited therapeutic options. Recently, extracellular vesicles derived from mesenchymal stem cells (MSC-EVs) have shown great potential in tissue regeneration and repair, offering a new avenue for the treatment of DKD. The purpose of this study is to explore the function and mechanism of action of EVs derived from human umbilical cord MSCs (hucMSC-EVs) in the development of DKD. Our findings show that under high-glucose (HG) conditions, miR-146b-5p is highly expressed in glomerular mesangial cells, downregulating the target protein Merlin, which promotes the activation of the YAP signaling pathway and induces mesangial cell fibrotic-like changes, leading to a significant deposition of collagen and interstitial fibrosis in the kidney. In vivo and in vitro experimental findings reveal that hucMSC-EVs can significantly inhibit miR-146b-5p, upregulate Merlin expression, prevent the nuclear translocation of YAP, improve renal function, and reduce collagen deposition, demonstrating a significant antifibrotic effect. The findings of this study emphasize the central role of the miR-146b-5p/Merlin/YAP axis in hucMSC-EVs-mediated inhibition of renal fibrosis and highlight the potential of MSC-EVs as a targeted nanotherapeutic strategy for DKD.

Ultraviolet (UV) radiation induces skin damage primarily through oxidative stress and excessive inflammation. Exosomes derived from mesenchymal stem cells have emerged as promising therapeutic agents for tissue repair. Here, we investigated the protective effects of human umbilical cord mesenchymal stem cell-derived exosomes (HuMSC-Exos) on UVB-induced skin injury in HaCaTs and C57BL/6 mice. HuMSC-Exos significantly reduced reactive oxygen species (ROS) levels, suppressed proinflammatory cytokines (IL-1β, TNF-α, and IL-6), and improved cell migration. Mechanistically, HuMSC-Exos inhibited Keap1, enhanced both total and phosphorylated Nrf2 expression, promoted its nuclear translocation, and upregulated antioxidant genes (HMOX1, NQO1, CAT, and SOD2). miR-200a-3p in HuMSC-Exos mediated these effects by targeting Keap1. Furthermore, preliminary data suggested that HuMSC-Exos also attenuate inflammatory responses via the NF-κB pathway. In vivo, HuMSC-Exos attenuated UVB-induced skin injury and inflammation by activating the Nrf2 signaling cascade. Collectively, our findings reveal a novel protective mechanism and highlight the therapeutic potential of HuMSC-Exos in mitigating UV-induced skin damage by modulating oxidative stress and inflammation.

Intervertebral disc degeneration (IVDD), a major contributor to chronic low back pain (LBP), involves progressive extracellular matrix (ECM) degradation and limited self-repair. Current therapies alleviate symptoms but fail to halt degeneration, driving interest in endogenous stem cell-based regeneration. Endogenous stem/progenitor cells within disc niches exhibit regenerative potential through ECM synthesis, anti-inflammatory signaling, and exosomal miRNA-mediated repair. Preclinical studies highlight mesenchymal stem cell (MSC) transplantation and reprogramed induced pluripotent stem cells (iPSCs) in restoring disc hydration and reducing pain, while early clinical trials report symptomatic relief (e.g., 70% pain reduction) but incomplete structural recovery. Challenges include the disc's hostile microenvironment (hypoxia and nutrient deprivation), age-related depletion of endogenous stem/progenitor cells, and impaired cell homing under mechanical stress. Emerging strategies target epigenetic modulation, biomimetic scaffolds, and combination therapies to enhance cell survival and integration. Despite promising preclinical outcomes, clinical translation requires overcoming microenvironmental barriers and refining delivery systems. Future efforts should prioritize large-animal validation and biomarker-guided approaches to bridge the gap between experimental success and therapeutic application.

Objective: To evaluate the impact of exosomes derived from rat bone marrow mesenchymal stem cells (BM-MSCs) on the malignant properties of human colorectal cancer stem cells (CRC-CSCs) and the underlying mechanism involving epithelial-mesenchymal transition (EMT).

Methods: Exosomes were isolated and characterized from rat BM-MSCs. Human CRC-CSCs were enriched from HCT116 cells and subsequently treated with the exosomes. Cellular functions, including proliferation, apoptosis, cell cycle progression, migration, and invasion, were assessed using cell counting kit-8 (CCK-8), colony formation, flow cytometry, and Transwell assays, respectively. In vivo tumorigenicity and lung metastasis were evaluated using a xenograft mouse model. Expression levels of EMT markers (E-cadherin, N-cadherin, and Vimentin) were analyzed by western blot, qPCR, and immunofluorescence.

Results: BM-MSCs-derived exosomes were efficiently internalized by HCT116-CSCs. In vitro, exosome treatment significantly enhanced cell proliferation, migration, invasion, and cell cycle progression, while suppressing apoptosis. In vivo, exosomes promoted tumor growth and lung metastasis. Mechanistically, exosome exposure induced EMT, as evidenced by decreased E-cadherin expression and increased expression of N-cadherin and vimentin in both in vitro and in vivo models.

Conclusion: Exosomes derived from rat BM-MSCs enhance the malignant phenotype and suppress apoptosis in human CRC-CSCs through the activation of the EMT pathway. These findings underscore the potential role of BM-MSC-derived exosomes in tumor microenvironment (TME) regulation and highlight their relevance as a potential therapeutic target.

Periodontal ligament stem cell spheroids are reportedly valuable for tissue regeneration; however, their application in vivo requires the use of a scaffold, which may raise safety concerns. We moulded human periodontal ligament mesenchymal stem cell (hPDLMSC) spheroids into blocks as a scaffold-free method for handling spheroids. We then examined the characteristics and osteogenic capabilities of hPDLMSC spheroid blocks in vitro and in vivo. First, the hPDLMSCs were seeded into microwell chips to form homogeneous spheroids, which were then seeded into net mould devices and cultured with rotary shaking to form hPDLMSC spheroid blocks. Next, real-time polymerase chain reaction (PCR) analysis, a live/dead assay and histological analysis were performed to investigate the properties of the hPDLMSC spheroid blocks. Finally, bone defects were created in mouse calvaria, and the defects were transplanted with hPDLMSC spheroid blocks; the osteogenic potential of the spheroid blocks was evaluated using three-dimensional (3D) micro-X-ray computed tomography (µCT) analysis and histological techniques. We identified that the expression levels of stemness markers and osteogenesis-related genes were higher in hPDLMSC spheroid blocks than in monolayer and spheroid-cultured hPDLMSCs. The live/dead assay and histological analysis revealed that there was almost no central necrosis in the hPDLMSC spheroid blocks, and hPDLMSC spheroid blocks formed nodules under osteogenic differentiation induction. Furthermore, the hPDLMSC spheroid block treatment group (without the use of scaffolds) exhibited both the nearly complete closure of the mouse calvarial bone defect and significantly increased bone microstructural parameters compared with the group in which hPDLMSC spheroids were transplanted in combination with scaffolds. Together, these findings indicate that hPDLMSC spheroid blocks possess excellent stemness and osteogenic potential, and may contribute to the establishment of novel scaffold-free therapies.