Epilepsy represents as one of the most prevalent and serious neurological disorders, with approximately one-third of patients resistant to conventional treatments. Current pharmacologic therapies primarily suppress seizures without targeting the underlying epileptogenesis, resulting in unchanged long-term prognosis. At the same time, epilepsy surgery remains underutilized due to the requirement for extensive evaluations and specialized expertise. Stem cell-based therapy has emerged as a promising avenue, offering the potential to address both the neurological and neuropsychiatric aspects. This review summarizes recent preclinical and clinical studies on stem cell therapy in epilepsy, with a focus on their mechanisms of action, including direct engraftment, restoration of normal neurogenesis, and immunomodulation via bystander effect. Advances in genetic engineering, extracellular vesicle-based therapy, and patient-derived brain organoids offer more personalized treatment strategies. Although stem cell therapy holds significant promise as a treatment for epilepsy, further extensive research is required to establish its clinical safety and efficacy.
INTRODUCTION:Human corneal stromal stem cells (hCSSCs), which exhibit mesenchymal stem cell (MSC) features, prevent fibrotic scarring and promote regeneration of injured stromal tissue, thereby restoring corneal clarity in mice. These therapeutic effects rely on hCSSCs' ability to respond to early inflammatory cues, suppress chronic neutrophil infiltration, and inhibit fibrosis. In this study, we examined the responses of hCSSCs to transient pro-inflammatory M1 stimulation and characterized the exosomes (Exo) they secreted. METHODS:We evaluated the expression of full-length protein-coding human TGFB3 mRNA molecules in purified Exo, and investigated Exo uptake, induction of human transforming growth factor β3 (hTGFβ3) protein expression, and the effects of different hTGFβ3/β1 ratios on myofibroblast generation from human corneal stromal keratocytes (CSKs) and stromal fibroblasts (SFs). The anti-scarring effect of hCSSC-Exo enriched in TGFB3 mRNA was assessed in a mouse model of acute corneal stromal injury. RESULTS:Our results showed that hCSSC-Exo contained TGFB3 mRNA transcripts, and their abundance increased following pro-inflammatory M1 stimulation. Exo uptake enhanced hTGFβ3 protein expression in hCSKs, and a higher hTGFβ3/β1 ratio in hCSKs and hSFs suppressed expression of α-smooth muscle actin and TGFβ1-induced myofibroblast differentiation. In vivo, topical treatment with M1-primed hCSSC-Exo reduced corneal opacity compared with sham-treated controls. Notably, hCSSC-Exo enriched with TGFB3 mRNA demonstrated greater scar-reducing and wound-healing efficacy. CONCLUSIONS:M1-primed hCSSC-Exo containing protein-coding TGFB3 mRNA significantly inhibited corneal opacity and promoted scar-free healing in injured mouse corneas. These findings support the anti-fibrotic and regenerative potential of hCSSC-Exo, which can be developed as a cell-free therapeutic strategy for corneal fibrosis and scarring.
BACKGROUND:Localized scleroderma (LoS) is a fibrotic skin disorder characterized by excessive collagen deposition, leading to functional and cosmetic impairments. Mesenchymal stromal cells (MSCs) offer therapeutic potential through immunomodulation and tissue repair, but their clinical use is limited by donor variability and replicative senescence. Induced pluripotent stem cell (iPSC)-derived MSCs (iMSCs) present a promising alternative owing to their scalable production and rejuvenated phenotype. METHODS:We generated adipose-derived iMSCs (AD-iMSCs) from reprogrammed adipose-derived stem cells (ADSCs) and compared their therapeutic efficacy to primary ADSCs in vitro and in vivo. Functional assays including transcriptomic profiling, conditioned medium (CM) effects on fibrosis markers (α-SMA, TGF-β/Smad), and angiogenesis were conducted. A bleomycin-induced murine LoS model was employed to assess AD-iMSC engraftment, collagen deposition, and microvascularization. RESULTS:AD-iMSCs exhibited rejuvenated-like phenotype, with reduced senescence markers and enhanced proliferative capacity. AD-iMSC-derived conditioned medium (AD-iMSCs-CM) outperformed ADSC-derived conditioned medium (ADSC-CM) in promoting angiogenesis in vitro. In vivo, AD-iMSCs demonstrated superior persistence and therapeutic efficacy, notably decreasing dermal thickness and collagen density while promoting vascularization. Mechanistically, AD-iMSCs more effectively suppressed the TGF-β/Smad2/3 signaling and extracellular matrix (ECM) remodeling pathways compared to ADSCs. CONCLUSION:In conclusion, our findings provide a robust preliminary proof-of-concept demonstrating that iPSC-mediated reprogramming yields iMSCs with rejuvenated-like phenotype and a potent anti-fibrotic secretome. These data offer a valuable molecular and functional rationale for the prospective development of iMSC-based platforms to circumvent the expansion limitations and donor variability inherent to primary MSC therapies for fibrotic skin disorders.
OBJECTIVE:To compare the therapeutic effects of exosomes from different cell sources and doses in rat knee osteoarthritis models using network meta-analysis, and to identify potential optimal strategies for preclinical optimization and clinical hypothesis generation. METHODS:A systematic search of PubMed, Web of Science, Embase, and Scopus identified 27 eligible randomized controlled trials (456 rats). Traditional random-effects meta-analysis and frequentist network meta-analysis were conducted. Treatments were ranked using SUCRA values. RESULTS:A total of 27 studies were included, comprising 456 rats. Traditional meta-analysis revealed that exosome therapy significantly reduced joint cartilage histopathological damage (reduced OARSI score: SMD = -3.78, 95% CI: -4.76, -2.80), promoted type II collagen synthesis (SMD = 3.41, 95% CI: 1.87, 4.95), and suppressed the expression of the inflammatory cytokine IL-1β (SMD = -3.26, 95% CI: -4.08, -2.44). Network meta-analysis further indicated that under high-dose conditions (≥100 μg), exosomes derived from human amniotic fluid stem cells (hAFSCs-Exo) and human umbilical cord mesenchymal stem cells (hUCMSCs-Exo) ranked highest in relative efficacy for improving the OARSI score. hUCMSCs-Exo also demonstrated the most potent anti-inflammatory effect by reducing IL-1β. Overall, exosome therapy exhibited a dose-related trend in efficacy, with high-dose transplantation showing higher probabilistic SUCRA rankings across most outcome measures. However, assessment of publication bias revealed marked asymmetry; after trim-and-fill correction, the effect size for type II collagen expression decreased from SMD = 3.41 to 1.26, suggesting that the magnitude of the treatment effect may have been overestimated. This overestimation reduces the certainty of the observed treatment effects. Despite the presence of publication bias and methodological limitations, the main conclusions remained statistically robust after trim-and-fill correction. CONCLUSION:Current evidence from rat models suggests that exosome therapy may substantially ameliorate the pathological progression of KOA, with exosomes derived from early developmental tissues, particularly hAFSCs-Exo and hUCMSCs-Exo, showing favorable chondroprotective and anti-inflammatory effects in the probabilistic ranking when administered at high doses. Nevertheless, the findings from publication bias and trim-and-fill analyses indicate that the existing effect sizes are likely overestimated. These results require confirmation in larger-scale, methodologically more rigorous preclinical studies.
Human stem cell (SC)-generated beta cell implants represent a potential cure for type 1 diabetes. Studies in rodents and patients showed that they can establish a glucose-regulated source of insulin in recipients with depleted pancreatic beta cell mass. Their therapeutic significance is, however, determined by their capacity to restore glycemic control by the human glucostat. It is so far unknown which implant characteristics are needed for this key endpoint. The present study addresses this question in mice. SC-derived preparations with a defined beta cell dose and alpha cell proportion were implanted in the epididymal fat pad of normoglycemic SCID/beige mice (mouse glucostat basal glycemia 166 mg/dL). At post-transplant week 20, 12/25 recipients reached glycemic control at the human glucostat (glycemia ≤ 90 mg/dL) following a time-dependent increase of basal and glucose-induced plasma human C-peptide and decrease of mouse C-peptide towards under detection limit. Formation of their functional human beta cell mass was preceded by formation of a human alpha cell mass that became the source for circulating glucagon and a contributor to glycemic control. The insulin content of the implants reached the values in the pancreas of control mice and were thus identified as in situ markers for implants that established the glycemic endpoint. Co-existence with a functional alpha cell mass places alpha cell formation as a potential additional component for achieving the goal. Our study also demonstrates the relevance of using normoglycemic mice to assess stem cell therapy protocols for beta cell replacement.
Rodent cell culture models have long underpinned research into nociceptive signaling; however, their limited recapitulation of human nociceptor biology has created a translational gap in analgesic development. While primary human sensory neurons are relevant, their use is hampered by scarcity, ethical constraints, donor variability, and difficulties in long-term culture. Furthermore, conventional cultures lack the compartmentalization needed to study sensory neuron processes and fail to recapitulate the synaptic connectivity between sensory and spinal cord neurons, limiting their translational relevance. To address these limitations, we utilized a microfluidic platform enabling compartmentalized culture of human induced pluripotent stem cell (hiPSC) derived sensory neurons (hiPSC-SNs) to study the function of their processes. We also demonstrate the feasibility of microfluidic co-cultures of hiPSC-SN with human iPSC-derived cortical excitatory neurons (hiPSC-CNs) as a basis for future development of models for sensory-to-CNs communication circuit. Using optimized protocols, we maintained stable microfluidic cultures and confirmed expression of pain-relevant sodium channels (Nav1.7, Nav1.8) in hiPSC-SN in both mono- and co-culture configurations. Leveraging this compartmentalized platform, we demonstrate that pharmacological blockade of Nav1.7 and Nav1.8 inhibits signal propagation along sensory neuron processes. We also demonstrate that growth factors modulate excitability of these processes. This functional validation underscores the platform's capability to investigate signal transmission along human sensory processes and demonstrates its potential for modelling more complex cellular interactions. Thus, we present a human iPSC-based microfluidic culture model that enables detailed study of sensory neuron processes and assessment of analgesics targeting nociceptive transmission, offering a significant advance toward analgesic drug discovery.
BACKGROUND AND AIMS:Cord blood infusion is an emerging treatment for cerebral palsy (CP) currently offered in the United States through clinical trials and an expanded access pathway. This study aimed to understand the barriers and facilitators influencing implementation of cord blood treatment for CP, to help inform future implementation efforts. METHODS:Semi-structured interviews were conducted with US professionals involved in the cord blood treatment program for CP at Duke University and a collaborator site. Data were analyzed using reflexive thematic analysis. RESULTS:From 16 interviews, 5 themes were identified capturing patterned barriers and enablers. Key facilitators included boundary-spanning clinical leadership, interprofessional coordination, dedicated resourcing, responsiveness to community demand, an established safety profile, and a strong research culture. Barriers included the need for cross-disciplinary expertise, uncertainty regarding efficacy evidence, funding and regulatory constraints, and system-level challenges. Participants described that many barriers were overcome to establish the program, while acknowledging that others may persist and are context dependent. CONCLUSIONS:Findings highlight that implementation of cord blood treatment for CP is feasible within specialized settings but contingent on local infrastructure, governance, and workforce capacity. Identified facilitators align with established implementation strategies and offer transferable insights, while underscoring the need for regulatory approval, ethical oversight, and system-level supports to enable equitable and sustainable access to emerging regenerative therapies for CP.
Diabetic vascular complications remain a major cause of morbidity and mortality, yet the molecular mechanisms underlying endothelial dysfunction in diabetes remain incompletely understood. Endothelial dysfunction is a key contributor to vascular pathology, and patient-derived induced pluripotent stem cell-derived endothelial cells (iPSC-ECs) provide a human platform to investigate disease-associated endothelial phenotypes under controlled conditions. Here, we identify the RNA-binding protein Quaking-7 (QKI-7) as a key regulatory factor associated with endothelial dysfunction in patient-derived iPSC-ECs. Using cells derived from diabetic and non-diabetic donors maintained under standard culture conditions, we demonstrate that elevated QKI-7 expression is associated with reduced expression of endothelial homeostatic genes (COL4A2, JUN, TMEM184A, and PPP1R15A) and impaired angiogenic capacity, including reduced tube formation. Importantly, these findings were further validated in three-dimensional blood vessel organoid models, supporting the relevance of QKI-7-associated endothelial phenotypes in a more physiologically complex vascular system. Connectivity mapping identified FDA-approved compounds, including simvastatin, halcinonide, and retinoic acid, as potential modulators of QKI-7-associated pathways. Functional validation in iPSC-ECs demonstrated that these compounds reduce QKI-7 expression and improve endothelial functional readouts. Together, these findings identify QKI-7 as a regulatory node associated with endothelial dysfunction in patient-derived iPSC-ECs and highlight the utility of human iPSC-based vascular models for identifying candidate therapeutic strategies. While these models capture endothelial phenotypes associated with diabetic donor origin, further studies in more complex in vivo systems will be required to establish causal relevance to vascular disease.
Platelet products are essential for preventing and treating bleeding in patients with thrombocytopenia. However, their short shelf life and reliance on voluntary blood donations pose significant challenges to maintaining a stable supply. To overcome these limitations, induced pluripotent stem cell-derived platelets (iPSC-PLTs) have emerged as a promising alternative. The clinical application of iPSC-PLTs succeeded in demonstrating safety in an autologous transfusion setting; however, allogeneic applications remain unexplored. Here, we report the world's first clinical evaluation of an allogeneic iPSC-PLT product. An immortalized megakaryocyte cell line (imMKCL) was established from an iPSC line by introducing 3 inducible genes-c-MYC, BMI1, and BCL-XL-and subsequently generating master and working cell banks. Using the working cell bank and turbulent flow bioreactors, an allogeneic iPSC-PLT product, MEG-002, was successfully produced with clinically relevant quality and yield. MEG-002 underwent comprehensive structural and functional characterization, including in vivo efficacy testing in rabbit models, which confirmed its functionality. Preclinical safety studies revealed no concerns. A clinical trial was conducted in accordance with ethical and regulatory standards in Japan. MEG-002 was infused into a patient with aplastic anemia at a dose of 6 × 1010 platelets. No adverse events were reported, and no clinically significant changes were observed in any assessments. Furthermore, a transient increase in platelet count and evidence of iPSC-PLT circulation were observed. Despite being descriptive observations from a single subject, these findings suggest the safety and potential efficacy of allogeneic iPSC-PLTs in humans. The clinical trial is registered with the Japan Registry of Clinical Trials (jRCT2053210068).
BACKGROUND:Human induced pluripotent stem cells (hiPSCs) are essential tools for disease modeling, drug testing, and regenerative medicine. These applications require differentiation of multiple cell lines in parallel, synchronized production of large numbers of hiPSCs and differentiated cells, and assessment of differentiation efficiency before proceeding to in-depth characterization through molecular and cellular assays. However, hiPSC culture protocols are often laborious manual processes, which affects reproducibility and makes high-throughput applications challenging. METHODS:We describe high-throughput hiPSC maintenance and amplification protocols that are weekend-free, scalable, and can be performed manually or automated. These optimized protocols enable scale-up of hiPSC production and differentiation to multiple cell types, including endothelial cells, microglia, and retinal pigment epithelial cells. RESULTS:We characterized the differentiated cells using molecular and cellular assays and demonstrated that cells generated with this approach represent accurate cell identities. CONCLUSION:The approaches described here enable high-throughput hiPSC applications and improve reproducibility and scalability.If you want, I can also tighten this further to better fit a strict 350-word journal limit or adapt the heading style to a specific journal.
Public cord blood (CB) banks face declining inventories worldwide, but the relative contributions of regulatory changes, declining birth rates, and infectious disease outbreaks remain unclear. We performed a retrospective analysis of 73 925 CB units submitted to a Korean public cord blood bank from 2006 to 2025, encompassing two regulatory transitions in total nucleated cell (TNC) criteria and three major outbreaks (H1N1, MERS, and COVID-19), using interrupted time series, correlation, and multivariable analyses. Of 73 925 submissions, 27 684 (37.4%) were banked. The 2011 TNC ≥ 8 × 108 criterion produced a moderate reduction in banking rate from 37.7% to 33.6%, while the 2021 TNC ≥ 11 × 108 revision caused an abrupt decline from 52.6% to 24.4%. H1N1 and MERS had minimal effects, whereas the COVID-19 period showed a sustained decline coinciding with the 2021 TNC revision. Birth decline correlated strongly with submissions (R2 = 0.94). Regulatory criteria changes were the primary driver of banking rate fluctuations, while declining births affected submission volume. Among three outbreaks, only the COVID-19 period overlapped with a sustained decline, likely reflecting concurrent regulatory change rather than the pandemic itself. CB quality and safety were maintained throughout all disruptions. These findings have direct translational implications for sustaining the global CB inventory as a critical source of hematopoietic stem cells for transplantation.
BACKGROUND:Osteoarthritis (OA) is a degenerative joint disease characterized by cartilage degradation, chondrocyte apoptosis, and inflammation. Mesenchymal stromal cells (MSCs) offer therapeutic potential, but their efficacy is hindered by oxidative stress and functional heterogeneity. We investigated whether PPARβ/δ priming enhances MSC therapeutic properties and explored the underlying mechanisms. METHODS:MSCs were primed with the PPARβ/δ agonist GW501516. In a collagenase-induced OA mouse model, we assessed cartilage integrity by confocal laser scanning microscopy and histological grading. In vitro, we evaluated the effects of primed MSCs on oxidative stress resistance, chondrocyte apoptosis, and immunomodulation using DNA fragmentation, co-culture assays, and RNA sequencing. RESULTS:PPARβ/δ priming significantly enhanced MSC resistance to oxidative stress, preserving their survival under H2O2-induced conditions. In co-culture experiments, primed MSCs exhibited a superior chondroprotective effect, reducing apoptosis in stressed chondrocytes. In vivo, primed MSCs improved cartilage volume, thickness, and integrity, lowering OA severity scores. RNA sequencing identified ANGPTL4 as a key mediator of these effects, as its silencing abolished MSC-mediated chondroprotection. CONCLUSION:PPARβ/δ priming enhances the therapeutic potential of MSCs by increasing their resistance to oxidative stress and anti-apoptotic effects on chondrocytes, mediated by ANGPTL4. These findings suggest a promising strategy for optimizing MSC-based therapies for OA.
Repairing bone defects resulting from trauma, infection, or tumor resection remains a significant challenge in clinical practice. Particularly in the oral and maxillofacial region, achieving predictable bone regeneration is especially difficult due to the complex anatomical structure and the critical need for optimal aesthetic and functional outcomes. A deeper understanding of the molecular mechanisms governing osteogenic differentiation is therefore essential to develop effective strategies for bone repair. Although creatine kinase B (Ckb) is known for its role in energy metabolism, its function in bone regeneration remains poorly understood. In this investigation, we found that Ckb is specifically expressed in jaw mesenchymal cells. Functional assays demonstrated that Ckb promoted osteogenic differentiation of bone marrow mesenchymal stem cells in vitro. Consistently, Ckb overexpression promoted alveolar bone healing in a mouse tooth-extraction model, while Ckb knockdown impaired bone regeneration. We discovered that Ckb localized to mitochondria and enhanced mitochondrial fission by increasing dynamin-related protein 1 (Drp1) Ser616 phosphorylation. The pro-osteogenic impact of Ckb was reduced by pharmacologically inhibiting mitochondrial fission with Mdivi-1. This result was replicated by Drp1 knockdown, indicating that Drp1 is essential for Ckb-mediated osteogenesis. Transcriptome sequencing identified αB-crystallin (Cryab) as a key downstream effector of Ckb, and co-immunoprecipitation confirmed a physical interaction between Ckb and Cryab. Crucially, Cryab overexpression rescued the osteogenic and mitochondrial dysfunction caused by Ckb knockdown. Collectively, our research reveals a unique Ckb-Cryab-Drp1 axis that controls mitochondrial dynamics to coordinate osteogenesis, offering a molecular basis for guiding MSC-based bone regeneration techniques.
INTRODUCTION:KATP-channel-related hyperinsulinism (KATPHI) is a rare genetic disorder of the pancreatic beta cells, manifesting as life-threatening hypoglycemia in neonates due to excessive insulin secretion. Management of the severe diffuse form of KATPHI currently lacks treatment options, as the first-line therapy octreotide is often insufficiently effective, necessitating radical pancreatectomy in many patients. METHODS:In this study, we differentiated stem cells carrying the KATPHI-causing mutation KCNJ11-/- to stem cell-derived islets (SC-islets) to develop new pharmaceutical therapies for KATPHI. We tested seven candidate molecules in vitro and the most effective ones in vivo in immunocompromised mice that were transplanted with the human KCNJ11-/- SC-islets. RESULTS:KCNJ11 -/- SC-islets inappropriately secreted 3.9 times more insulin in low glucose than KCNJ11+/+ controls. Transplanted KCNJ11-/- SC-islets caused persistent hyperinsulinemia and hypoglycemia to the recipient mice. We tested acyl-ghrelin identified in our in vitro experiments and its long-acting analogue relamorelin in these mice. Acyl-ghrelin and relamorelin alone increased fasting blood glucose. Combining relamorelin with octreotide increased blood glucose synergistically, ie, more than the sum of each drug alone, reverting hypoglycemia to normoglycemia while reducing the excessive insulin secretion. CONCLUSIONS:We show that ghrelin-receptor agonists have acute anti-hypoglycemic effects in a humanized mouse model of KATPHI, especially when combined with octreotide. Relamorelin has been tested in >650 diabetic adults with little side effects, leading us to propose relamorelin-octreotide combination for further development as a therapeutic candidate for severe KATPHI patients.
Long-term excessive fluoride ingestion may lead to skeletal fluorosis, a disease characterized by unclear pathogenesis and lack of effective cure. Bone marrow mesenchymal stem cell-derived extracellular vesicles (BMSCs-EVs) exert a crucial regulatory effect on bone remodeling and have broad clinical application prospects. Whether BMSCs-EVs can participate in the occurrence and development of skeletal fluorosis by transferring miRNAs is a scientific question worth exploring. In this study, miRNA sequencing was used to screen for differentially expressed miRNAs in fluoride-exposed rBMSCs-EVs (F-EVs). The top 15 differentially expressed miRNAs were subjected to target gene prediction, and GO and KEGG analyses as well as PPI network construction were performed. On this basis, miR-320-3p was selected for in-depth study. First, our data revealed that miR-320-3p was upregulated in fluoride-exposed osteoblasts and was involved in the enhancement of fluoride-induced osteogenic differentiation. Subsequently, a tracing experiment confirmed that rBMSCs-EVs and their miR-320-3p could be successfully taken up by fluoride-exposed osteoblasts. Then, elevated levels of miR-320-3p in F-EVs could further promote the enhancement of fluoride-induced osteogenic differentiation, while EVs with inhibited miR-320-3p exerted a significant inhibitory effect. Mechanism studies revealed that under fluoride exposure, rBMSCs-EVs could regulate Igf1r/ERK1/2/MAPK by transferring miR-320-3p, thereby affecting osteogenic differentiation. This study is the first to elucidate the pathogenesis of skeletal fluorosis from the rBMSCs-EVs miRNAs and provides new ideas for the clinical management of skeletal fluorosis.
Pluripotent stem cell (PSC)-based therapies hold the potential to unlock cures for numerous diseases, including, but not limited to, Parkinson's disease, macular degeneration, heart failure, type 1 diabetes, and cancer. Yet as protocols to differentiate PSCs into therapeutically useful cell types have progressed rapidly, immunological rejection remains a major barrier that may limit the widespread use of such PSC-based therapies. In recent years, strategies to genetically modify PSCs to prevent immunological rejection of the downstream cell product have become a point of emphasis. Here, we provide an immunological perspective on these strategies, discussing the breadth of rejection mechanisms that have been uncovered through decades of research and the relative simplicity of designing PSC immune evasion strategies to circumvent these mechanisms. We focus in particular on how these strategies apply to the treatment of type 1 diabetes.
The regeneration of a functional dentin-pulp complex represents a paramount objective in regenerative endodontics. However, current strategies reliant on scaffolds and growth factors are constrained by imprecise biological signaling and inefficient stem cell guidance. To address these limitations, this study aimed to develop a precise nanomaterial-based strategy. We investigated tetrahedral DNA nanostructures (TDNs) as bioactive nanocarriers to enhance the odontogenic differentiation of human dental pulp stem cell-derived induced pluripotent stem cells (hDPSC-iPSCs) within a gelatin methacryloyl (GelMA) hydrogel system. hDPSCs were isolated from young patients' teeth, reprogrammed into iPSCs, and characterized. TDNs were synthesized, characterized, and shown to be efficiently internalized by cells. In vitro, TDN treatment significantly upregulated key odontogenic markers (DSPP and DMP-1), promoted proliferation, and enhanced migration of hDPSC-iPSCs. For in vivo assessment, TDN-pretreated hDPSC-iPSCs encapsulated in GelMA were implanted into a tooth root canal regeneration model in nude mice. The TDN group regenerated well-structured, dentin-pulp-like tissues exhibiting strong dentin sialophosphoprotein (DSPP) expression, indicative of odontogenic differentiation. Collectively, these findings demonstrate that TDNs potently direct hDPSC-iPSCs toward an odontogenic lineage and support pulp-like tissue formation within a hydrogel niche. The integrated TDN-cell-GelMA platform presents a novel, precise, and minimally invasive strategy for early-stage pulp-like regeneration, with promising potential for clinical translation in regenerative endodontics.
Type 1 diabetes (T1D) is a chronic autoimmune disease characterized by the destruction of pancreatic β cells, leading to lifelong insulin dependence and an increased risk of severe complications. Three-dimensional stem cells (3D SCs) culture systems have emerged as a superior alternative by more accurately mimicking the in vivo microenvironment and enhancing stemness maintenance, regenerative efficiency, and paracrine secretion. However, studies exploring the application of 3D SCs in T1D remain limited. Here, we developed a novel serum- and cytokine-free orbital-shaking system. It enables efficient and large-scale reprogramming of somatic cells into 3D embryonic-like stem cell spheroids (Sph-Es) characterized by robust pluripotency and improved safety. To enhance therapeutic utility, Sph-Es were irradiated and transduced with INS-expressing adenoviral vectors to generate Sph-R-Ins, allowing transient insulin production without permanent genomic modification. In STZ-induced T1D mice, Sph-R-Ins improved glycemic control and glucose tolerance and increased mouse insulin and C-peptide responses, indicating improved endogenous islet function. Donor-cell tracking analyses showed no pancreatic engraftment, supporting an indirect mode of action. Additional transcriptomic, immunological, and ex vivo studies indicated that the therapeutic benefit was accompanied by ECM-related signaling changes, reduced inflammatory infiltration, enhanced M2 macrophage polarization and Treg-associated immune regulation, improved metabolic signaling, and spheroid-derived paracrine support of islet function. Together, these findings establish a mechanically guided 3D stem cell-gene therapy platform with both endocrine and immunometabolic benefits in T1D.
BACKGROUND/PURPOSE:Mesenchymal stem cell (MSC) therapy offers promise for treating autoimmune diseases due to its strong immunomodulatory effects. We investigated the long-term safety of a single intravenous injection with human umbilical cord blood-derived (hUCB)-MSCs in patients with rheumatoid arthritis (RA). METHODS:Patients with RA who met the 2010 ACR/EULAR classification criteria and received a single intravenous infusion of hUCB-MSCs (2.5 × 107, 5.0 × 107, 1.0 × 108 cells) in a phase I trial (NCT02221258) entered this 5-year observational pilot study. Safety assessments were performed at 3, 6, and 12 months after infusion and annually thereafter. Safety endpoints included overall adverse events (AEs), serious adverse events (SAEs), and AEs of special interest. RESULTS:Nine patients were treated. The most common AEs were osteoarthritis (44.4%) and nasopharyngitis (44.4%). SAEs occurred in 5 patients (55.6%); a serious infection (cellulitis) occurred in 1 patient in the 1.0 × 108 group and resolved after treatment. Benign ovarian and breast tumors were reported in 2 patients 3 years post-infusion. No deaths, thromboembolism, or malignancies occurred during the follow-up period. Laboratory findings remained stable except for 1 case each of transient hypertriglyceridemia and mild eosinophilia. While DAS28 improved markedly by 3-6 months, disease activity gradually increased over 5 years, suggesting waning efficacy after a single infusion. CONCLUSION:The long-term safety profile of a single dose of intravenous hUCB-MSC in patients with RA appears acceptable; however, repeated-dose regimens may be needed for sustained disease control and further safety evaluation.
BACKGROUND:Red blood cell (RBC) transfusions are essential for treating various medical conditions, but global demand is difficult to meet due to a dwindling donor pool and compatibility issues. Pluripotent stem cells (PSCs) offer a promising alternative of blood dependent on volunteer donors for RBC production, and dogs serve as an excellent model for translational research due to their physiological and genetic similarities to humans. METHODS:Canine induced pluripotent stem cells (ciPSCs) were differentiated toward hematopoietic and erythroid lineages. Differentiated cells were evaluated for hematopoietic marker expression, hemoglobinization, colony-forming capacity, enucleation, and hemoglobin gene expression. Glycophorin A (GYPA)-enhanced green fluorescent protein (EGFP) reporter ciPSC lines were generated using clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9-mediated genome editing to visualize GYPA expression during differentiation. RESULTS:This study introduces a protocol for RBC differentiation using ciPSCs. We achieved generation of hemoglobinized RBCs, progressing through polychromatic and orthochromatic erythroblast-like stages. CiPSC-derived hematopoietic cells/RBCs were confirmed to have immature characteristics as determined by limited colony-forming capacities, low enucleation, and embryonic and fetal hemoglobin gene expression. Additionally, we created GYPA-EGFP reporter ciPSC lines using CRISPR-Cas9-mediated genome editing, enabling real-time visualization of GYPA expression. This innovation confirmed GYPA as a viable surface marker for ciPSC-derived RBCs. CONCLUSION:Our findings mark an initial step toward establishing a canine PSC-based erythroid differentiation system, providing a foundation for future improvements and exploration of applications for canine PSC-derived RBCs.
