Stem Cell Research & Therapy最新文献

Background: Autologous fat grafting (AFG) often needs multiple sessions due to low volume retention. Young adipose tissue demonstrates a more pronounced therapeutic effect; thus, the cryopreservation of adipose tissue of young origin is particularly crucial. This study investigated the protective effect of a new cryopreservation solution combining trehalose, glycerol, and metformin on adipose tissue.

Methods: This study initially examined the effect of various concentrations of metformin (0, 1, 2, 4, and 8 mM) on oxidative damage in adipose tissue to identify the optimal concentration. Subsequently, 1.5 mL of fresh human adipose tissue was subjected to freezing using trehalose + glycerol (TG group), trehalose + glycerol + metformin (TGM group), and the common cryoprotectant dimethyl sulfoxide (DMSO) + fetal bovine serum (FBS) (DF group). Samples were cryopreserved in liquid nitrogen for 2 weeks. After thawing, 1 mL of adipose tissue from each group was transplanted subcutaneously into the backs of nude mice. The cryoprotective effects on adipose tissue viability were evaluated during transplantation one month after transplantation.

Results: The 2 mM concentration of metformin exhibited the lowest reactive oxygen species (ROS) level (29.20 ± 1.73) compared to other concentrations (P < 0.05). Cell proliferation and migration assays also supported the superior performance of the 2 mM concentration. Apoptotic analyses of stromal vascular fraction (SVF) cells showed the lowest levels in the 2 mM group. Compared to other cryopreservation groups, the adipose tissue in the TGM group closely resembled fresh adipose tissue in terms of gross structure and histological characteristics, with the lowest apoptosis rate of SVF cells. In vivo analysis revealed the highest tissue retention rate in the TGM group, with histological examination indicating robust structural integrity.

Conclusion: The TGM cryopreservation solution, containing metformin, greatly preserves adipose tissue, reduces apoptosis, and improves tissue retention rates. This solution was non-toxic and safe, making it well-suited for tissue cryopreservation in clinical settings.

Background: Methicillin-resistant Staphylococcus aureus (MRSA) is still a growing concern in the field of antimicrobial resistance due to its resistance to conventional antibiotics and its association with high mortality rates. Mesenchymal stromal cells (MSCs) have been shown as a promising and attractive alternative treatment for bacterial infections, due to their antibacterial properties and potential to bypass traditional resistance mechanisms. This study aims to shed light on the antibacterial potential of adipose-derived mesenchymal stromal cell (AD-MSC) secretome against clinical isolates of Staphylococcus spp., including MRSA strains.

Methods: Using the Kirby-Bauer disk diffusion method, broth microdilution assays, and colony-forming unit (CFU) counting, the antibacterial activity of AD-MSC secretome was assessed. These tests were first conducted on Staphylococcus (S.) aureus ATCC 25923, then on 73 clinical isolates including MRSA strains. Further molecular analysis was performed to identify resistant genes in MRSA isolates.

Results: The AD-MSC secretome demonstrated significant antibacterial activity against S. aureus ATCC with a 32 mm inhibition zone. 96% of the collected staphylococcal clinical isolates showed susceptibility to the secretome with 87.5% inhibition observed in MRSA isolates, along with 100% in MSSA, MSSE, and MRSE strains. Molecular analysis revealed that MRSA strains resistant to the secretome harbored mecA, ermA, and ermB genes. Additionally, the mecA-negative MRSA strains remained susceptible to the secretome, suggesting alternative resistance mechanisms.

Conclusion: These findings emphasize the ability of AD-MSCs secretome as a promising alternative for treating antibiotic-resistant infections, with potential applications in combating MRSA. However, further research is required to explore its clinical applications as a complementary or standalone therapy for resistant infections.

Background: Mesenchymal stem cells have great potential for repairing articular cartilage and treating knee osteoarthritis (KOA). Nonetheless, little is known about the efficacy of human adipose-derived mesenchymal stem cells (haMSCs) for KOA in large animal models.

Methods: This study evaluated the therapeutic efficacy of haMSCs in knee articular cartilage repair in a sheep model of KOA. haMSCs were isolated, cultured, and characterized. KOA was surgically induced by anterior cruciate ligament transection and medial meniscectomy, followed by intra-articular injection of saline (negative control group) or haMSCs (haMSC group) into the right knee joint at 6 and 9 weeks after surgery. Sheep were sacrificed 21 weeks after surgery, and samples (whole knee joints, femoral condyles, and tibias) were collected, processed, and analyzed. Changes in knee articular cartilage were assessed by magnetic resonance imaging, micro-computed tomography, macroscopic analysis, histology, and immunohistochemistry.

Results: KOA caused the degeneration of the medial femoral condyle in the sheep model of KOA. Conversely, haMSCs repaired chondral defects and increased the thickness of knee articular cartilage.

Conclusions: These data suggest that the intra-articular injection of haMSCs can effectively repair articular cartilage defects in the knee.

Background: Interaction between mesenchymal stem cells (MSCs) and oral squamous cell carcinoma (OSCC) cells plays a major role in OSCC progression. However, little is known about adipogenic differentiation alteration in OSCC-derived MSCs (OSCC-MSCs) and how these alterations affect OSCC growth.

Methods: MSCs were successfully isolated and cultured from normal gingival tissue, OSCC peritumoral tissue, and OSCC tissue. This included gingiva-derived MSCs (GMSCs), OSCC adjacent noncancerous tissues-derived MSCs (OSCCN-MSCs), and OSCC-MSCs. The adipogenic and osteogenic differentiation capabilities of these cells were evaluated using Oil Red O and Alizarin Red S staining, respectively. OSCC cells were then co-cultured with either OSCC-MSCs or GMSCs to assess the impact on OSCC cell proliferation and migration. Subcutaneous xenograft experiments were conducted in BALB/c-nu mice to further investigate the effects in vivo. Additionally, immunohistochemical staining was performed on clinical samples to determine the expression levels of fatty acid synthase (FASN) and the proliferation marker Ki67.

Results: OSCC-MSCs exhibited enhanced adipogenic differentiation and reduced osteogenic differentiation compared to GMSCs. OSCC-MSCs significantly increased the proliferation and migration of OSCC cells relative to GMSCs and promoted tumor growth in mouse xenografts. Lipid droplet accumulation in the stroma was significantly more pronounced in OSCC + OSCC-MSCs xenografts compared to OSCC + GMSCs xenografts. Free fatty acids (FFAs) levels were elevated in OSCC tissues compared to normal gingival tissues. Moreover, OSCC-MSCs consistently secreted higher levels of FFAs in condition medium than GMSCs. Knockdown of FASN in OSCC-MSCs reduced their adipogenic potential and inhibited their ability to promote OSCC cell proliferation and migration. Clinical sample analysis confirmed higher FASN expression in OSCC stroma, correlating with larger tumor size and increased Ki67 expression in cancer tissues, and was associated with poorer overall survival.

Conclusions: OSCC-MSCs promoted OSCC proliferation and migration by upregulating FASN expression and facilitating FFAs secretion. Our results provide new insight into the mechanism of OSCC progression and suggest that the FASN of OSCC-MSCs may be potential targets of OSCC in the future.

Despite significant strides in medical treatments and surgical procedures for cardiovascular diseases, these conditions continue to be a major global health concern. The persistent need for innovative therapeutic approaches to mend damaged heart tissue highlights the complexity and urgency of this medical challenge. In recent years, stem cells have emerged as a promising tool for tissue regeneration, but challenges such as graft rejection and tumor formation have limited their clinical application. Exosomes, extracellular vesicles containing a diverse array of biomolecules, have garnered significant attention for their potential in regenerative medicine. The cardioprotective and reparative properties of mesenchymal stem cell-derived exosomes hold promise for the treatment of heart diseases. These exosomes can modulate various cellular processes, including angiogenesis, apoptosis, and inflammation, thereby enhancing cardiac function. Despite the growing interest, there remains a lack of comprehensive reviews synthesizing the molecular mechanisms, preclinical, and clinical evidence related to the specific role of MSC-derived exosomes in cardiac therapies. This review aims to fill that gap by exploring the potential of MSC-derived exosomes as a therapeutic strategy for cardiac diseases. This review explores the potential of mesenchymal stem cell-derived exosomes as a therapeutic strategy for cardiac diseases. We discuss the molecular mechanisms underlying their cardioprotective effects, summarize preclinical and clinical studies investigating their efficacy, and address the challenges and future perspectives of exosome-based therapies. The collective evidence suggests that MSC-derived exosomes hold promise as a novel and effective therapeutic approach for cardiac diseases.

Background and aim: Human dental pulp stem cells (hDPSCs) constitute a promising alternative for central nervous system (CNS) cell therapy. Unlike other human stem cells, hDPSCs can be differentiated, without genetic modification, to neural cells that secrete neuroprotective factors. However, a better understanding of their real capacity to give rise to functional neurons and integrate into synaptic networks is still needed. For that, ex vivo differentiation protocols must be refined, especially to avoid the use of fetal animal serum. The aim of our study is to improve existing differentiation protocols of hDPSCs into neuron-like cells.

Methods: We compared the effects of the (1) absence or presence of fetal serum during the initial expansion phase as a step prior to switching cultures to neurodifferentiation media. We (2) improved hDPSC neurodifferentiation by adding retinoic acid (RA) and potassium chloride (KCl) pulses for 21 or 60 days and characterized the results by immunofluorescence, digital morphometric analysis, RT-qPCR and electrophysiology.

Results: We found that neural markers like Nestin, GFAP, S100β and p75^NTR were expressed differently in neurodifferentiated hDPSC cultures depending on the presence or absence of serum during the initial cell expansion phase. In addition, hDPSCs previously grown as spheroids in serum-free medium exhibited in vitro expression of neuronal markers such as doublecortin (DCX), neuronal nuclear antigen (NeuN), Ankyrin-G and MAP2 after neurodifferentiation. Presynaptic vGLUT2, Synapsin-I, and excitatory glutamatergic and inhibitory GABAergic postsynaptic scaffold proteins and receptor subunits were also present in these neurodifferentiated hDPSCs. Treatment with KCl and RA increased the amount of both voltage-gated Na⁺ and K⁺ channel subunits in neurodifferentiated hDPSCs at the transcript level. Consistently, these cells displayed voltage-dependent K⁺ and TTX-sensitive Na⁺ currents as well as spontaneous electrophysiological activity and repetitive neuronal action potentials with a full baseline potential recovery.

Conclusion: Our study demonstrates that hDPSCs can be differentiated to neuronal-like cells that display functional excitability and thus evidence the potential of these easily accessible human stem cells for nerve tissue engineering. These results highlight the importance of choosing an appropriate culture protocol to successfully neurodifferentiate hDPSCs.

Background: Despite many years of investigation into mesenchymal stem cells (MSCs) and their potential for treating inflammatory conditions such as COVID-19, clinical outcomes remain variable due to factors like donor variability, different tissue sources, and diversity within MSC populations. Variations in MSCs' secretory and proliferation profiles, and their proteomic and transcriptional characteristics significantly influence their therapeutic potency, highlighting the need for enhanced characterization methods to better predict their efficacy. This study aimed to evaluate the biological characteristics of MSCs from different tissue origins, selecting the most promising line for further validation in a K18-hACE2 mouse model of SARS-CoV-2 infection.

Methods: We studied nine MSC lines sourced from either bone marrow (hBMMSC), dental pulp (hDPMSC), or umbilical cord tissue (hUCMSC). The cells were assessed for their proliferative capacity, immunophenotype, trilineage differentiation, proteomic profile, and in vitro immunomodulatory potential by co-culture with activated lymphocytes. The most promising MSC line was selected for further experimental validation using the K18-hACE2 mouse model of SARS-CoV-2 infection.

Results: The analyzed cells met the minimum criteria for defining MSCs, including the expression of surface molecules and differentiation capacity, showing genetic stability and proliferative potential. Proteomic analysis revealed distinct protein profiles that correlate with the tissue origin of MSCs. The immunomodulatory response exhibited variability, lacking a discernible pattern associated with their origin. In co-culture assays with lymphocytes activated with anti-CD3/CD28 beads, all MSC lines demonstrated the ability to inhibit TNF-α, to induce TGF-β and Indoleamine 2,3-dioxygenase (IDO), with varying degrees of inhibition observed for IFN-γ and IL-6, or induction of IL-10 expression. A module of proteins was found to statistically correlate with the potency of IL-6 modulation, leading to the selection of one of the hUCMSCs as the most promising line. Administration of hUCMSC to SARS-CoV-2-infected K18 mice expressing hACE2 was effective in improving lung histology and modulating of a panel of cytokines.

Conclusions: Our study assessed MSCs derived from various tissues, uncovering significant variability in their characteristics and immunomodulatory capacities. Particularly, hUCMSCs demonstrated potential in mitigating lung pathology in a SARS-CoV-2 infection model, suggesting their promising therapeutic efficacy.

Background: Culture medium enriched with Knockout serum replacement (KSR) can produce in vitro mouse sperm, but it is inefficient, strain-specific and contains bovine products, which limits its use in the human clinic. The study aimed to optimize the culture medium for testicular tissue by using plasma rich in growth factors (PRGF) as a serum supplement, addressing the limitations of KSR.

Methods: Immature testicular tissues from NMRI mice were cultured for 14 days to identify the optimal PRGF concentration using histological analysis and tubular integrity scoring. Subsequently, tissues were cultured for 42 days with the optimal PRGF concentration and compared to a control group with 10% KSR, followed by evaluation through histological, tubular integrity, and immunofluorescence assays.

Results: After 14 days, 5% PRGF media significantly preserved tubule integrity better than 10% and 20% PRGF, performing similarly to 10% KSR. However, after 42 days, the integrity scoring revealed significantly a higher percentage of well-preserved tubules in 5% PRGF compared to 10% KSR. Additionally, only PRGF supported spermatogenesis to the production of flagellated sperm. Real-time PCR analysis revealed that transcript levels of Plzf, Tekt1, and Tnp1 were significantly elevated in 5% PRGF compared to 10% KSR. Immunofluorescence and quantitative analysis confirmed enhanced spermatogenesis progression in 5% PRGF media, with significantly increased numbers of PLZF + spermatogonia, SYCP3 + spermatocytes, ACRBP + spermatids, and Ki67 + proliferating cells per tubule compared to 10% KSR. Moreover, 5% PRGF showed a significantly lower mean fluorescence intensity of the pro-apoptotic marker Bax, with no significant difference in the anti-apoptotic marker Bcl-2 compared to KSR.

Conclusions: The findings suggest that 5%PRGF is a viable alternative to KSR in mouse testicular tissue cultures, promoting structural integrity and spermatogenesis up to the production of flagellated sperm. The results highlight PRGF's potential to improve culture media for in vitro sperm production, suggesting promising avenues for future human research.

Background: Alzheimer's disease (AD) is a progressive neurodegenerative condition affecting around 50 million people worldwide. Bone marrow-derived mesenchymal stem cells (BMMSCs) have emerged as a promising source for cellular therapy due to their ability to differentiate into multiple cell types and their paracrine effects. However, the direct injection of BMMSCs can lead to potential unpredictable impairments, prompting a renewed interest in their paracrine effects for AD treatment. The specific mechanism and central role of cytokines in this process have not been fully elucidated.

Methods: Mouse BMMSCs were isolated, validated, and then transplanted intracerebrally into APP/PS1 female mice. The behavioral tests, including open-field test, novel object recognition test, and Morris water maze were performed, followed by β-amyloidosis plaque and neuron apoptosis analyses. Then the tissue RNA sequencing and mBMMSC cytokine analysis were performed. A cytokine antibody array for BMMSCs and the brain slice models were performed with AD model tissues were used to elucidate the molecular mechanisms. Finally, APP/PS1 mice were administrated with cytokine mixture for cognitive recovery.

Results: Our results demonstrated that BMMSCs significantly improved cognitive function, reduced beta-amyloid plaque deposition, and decreased apoptotic neurons through the activation of the AKT signaling pathway. Using a cytokine antibody array, we identified three highly expressed AKT pathway regulated neuroprotective factors in BMMSCs: IGF1, VEGF, and Periostin2. These cytokines were found to upregulate inhibitors of apoptosis family proteins (IAPs) and suppress Caspase-3 activity in brain slices induced with beta amyloidosis (Aβ), okadaic acid (OA), and lipopolysaccharide (LPS). When injection of this cytokine mixture to APP/PS1 mice also resulted in a mitigation of cognitive impairment.

Conclusions: These findings suggest that the secretory factors IGF1, VEGF, and Periostin2 derived from BMMSCs play a crucial role in neuroprotection by modulating the AKT/IAPs pathway to restore neuronal function. These cytokine sets could be a potential therapeutic strategy for AD and lay the groundwork for promising clinical applications.

Background: Understanding how enteric neural crest cells (ENCCs) differentiate into neurons is crucial for neurogenesis therapy and gastrointestinal disease research. This study explores how magnesium ions regulate the glycolytic pathway to enhance ENCCs differentiation into neurons.

Materials and methods: We used polymerase chain reaction, western blot, immunofluorescence, and multielectrode array techniques to assess magnesium ions' impact on ENCCs differentiation. Non-targeted metabolomic sequencing, cellular acidification rate, oxygen consumption, and western blot analyzed sugar metabolism changes. D-glucose-¹³C6 isotope tracing identified key glucose flux changes. Surface plasmon resonance was used to detect the binding affinity of magnesium ions with key glycolysis genes. The elastic modulus of the hydrogel was measured using a universal testing machine, while pore size and porosity were assessed with scanning electron microscopy. Swelling ratios were determined using gravimetric analysis. In vivo, ENCCs in hydrogels were transplanted into renal capsule and subcutaneously, and magnesium ions' effects on ENCCs differentiation were evaluated.

Results: Magnesium ions increased glycolysis levels during ENCCs differentiation into neurons, along with significant upregulation of neuronal markers β-Tubulin and ubiquitin C-terminal hydrolase L1, and enhanced functional neuronal properties. D-glucose-¹³C6 tracing results showed increased carbon flux in the glycolytic pathway after magnesium supplementation. The binding affinity of magnesium ions with the glycolytic key enzyme 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3 was found to be 1.08 μM. Inhibiting glycolysis suppressed ENCCs differentiation into neurons, emphasizing its crucial role. The double-cross-linked hydrogel gelatin methacryloyl-alginate (gelMA-ALMA), cross-linked with magnesium ions, showed promise in enhancing ENCCs differentiation in vivo without causing systemic hypermagnesemia.

Conclusion: Magnesium ions promote ENCCs differentiation into neurons by activating the Warburg effect. The GelMA-ALMA hydrogel serves as an effective localized magnesium delivery system, supporting neuronal differentiation in vivo.