Purpose: Human amniotic fluid stem cells (hAFSCs) have shown significant regenerative potential in treating hair loss, wound healing, and tissue repair. This study aims to evaluate the effects of human amniotic fluid (hAF) on hair follicle (HF) regeneration and immune system modulation.

Materials and Methods: The hAF used was pooled, acellular, and gamma-irradiated to standardize its contents and enhance its stability. Both irradiated (FAFI) and non-irradiated (FAF) hAF were assessed for their efficacy and safety in promoting hair growth and modulating immune responses in a rat model of hair loss. The study examined HF regeneration, transition to the anagen phase, and macrophage polarization from the pro-inflammatory M1 phenotype to the anti-inflammatory M2 phenotype.

Results: Both FAF and FAFI treatments significantly increased HF density, with FAFI exhibiting enhanced effects. Histological analysis demonstrated improved HF regeneration, increased M2 macrophages, and reduced collagen fiber deposition in treated areas. Gamma irradiation likely improved the efficacy of FAFI by stabilizing active components and inhibiting protease activity.

Conclusions: Irradiated hAF is a safe and effective therapeutic candidate for alopecia and HF growth disorders. These findings support further evaluation of hAF in clinical trials to validate its potential for hair regeneration therapies.

Mesenchymal stem cells (MSCs) have come up as a potential remedy for treatment of various diseases thanks to their regenerative abilities. However, MSC-based therapies face challenges like reduced cell survival and functionality after transplantation. Preconditioning, particularly with hypoxia-mimicking agents like cobalt chloride (CoCl2), has been explored to enhance MSCs' effectiveness. This study aims to evaluate MSC survival, migration, and therapeutic outcomes at the CoCI2-preconditioning and post-preconditioning stages. Human umbilical cord-MSCs were treated with 100 µM CoCI2 with/out serum for 24-hours, and then passaged and planted in corresponding culture conditions without CoCI2, these two consecutive passages were named as the preconditioning and post-preconditioning stages, respectively. In each stage, total protein concentrations, total antioxidant and total oxidant status (TAS/TOS) of the conditioned media derived from the cells were investigated with bicinchoninic acid assay and TAS/TOS kits, respectively. The proliferation rates, migratory capacities, cellular senescence, expression levels of hypoxia-inducible factor1-α (HIF1-α), Ki-67, active caspase-3 and beclin-1 proteins and ultrastructures of the cells were evaluated by 3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide test, wound healing assay, β-galactosidase-activity assessment, immunocytochemistry and transmission electron microscopy, respectively. Our results indicated that preconditioning MSCs with CoCl2 did not significantly enhance their proliferation, migration, or secretory abilities. However, it increased antioxidant capacity and along with normalization of senescence-status post-preconditioning, possibly by shifting energy metabolism from oxidative-phosphorylation to glycolysis through the upregulation of the HIF1-α signalling pathway. These findings indicate that CoCl2 preconditioning could be an effective approach to boost the therapeutic potential of MSCs, especially in enhancing their survival and functionality after transplantation.

Acute kidney injury (AKI) is a life-threating syndrome characterized by sudden loss of kidney function, and its management is challenging and often suboptimal. Mesenchymal stem cells (MSCs) have shown promise in AKI therapy in pre-clinical and clinical trials; however, their clinical application still faces many challenges. MSC-derived small extracellular vesicles (sEV) may help overcome these challenges. In the current study, we overexpressed Klotho in MSCs and then isolated Klotho-loaded sEV (Klotho-sEV) using anion-exchange chromatography. Klotho-sEV displayed characteristics comparable to those of sEV in terms of size, morphology, conventional markers, and biosafety, as well as a higher abundance of Klotho protein. In rhabdomyolysis-induced AKI, sEV showed preferential tropism in injured kidneys. We found significantly and stably accelerated renal recovery, mitigated functional and histological abnormalities, stimulated tubular cell proliferation, reduced injury and inflammatory marker expression, and restored endogenous Klotho loss in mice after the administration of Klotho-sEV. In addition, Klotho-sEV treatment activated the mTOR and MEK1/2 signaling pathways. Proteomics and small RNA sequencing analyses of sEV and Klotho-sEV revealed abundant proteins and miRNAs involved in anti-inflammation and reno-protection, and Klotho-sEV showed characteristics that were different from those of sEV. In conclusion, Klotho-sEV may be a promising cell-free strategy for the treatment of AKI.

Osteonecrosis of the femoral head (ONFH) is a refractory orthopedic disease that commonly affects young and middle-aged individuals. Long-term and high-dose use of glucocorticoids (GCs) is one of the main causes. Currently, the pathological mechanism of GCs-induced ONFH remains unclear, which poses difficulties for clinical prevention and treatment. This article focuses on reviewing the roles of gap junctions (GJs) and connexin 43 (Cx43) in GCs-induced ONFH. Under normal circumstances, cells in bone tissue form a network structure through GJs to maintain bone metabolic balance. However, GCs can obstruct the normal connections and signal transmission between bone tissue cells, leading to bone metabolic imbalance and triggering ONFH. As a key component of GJs in bone tissue, Cx43 is of great significance in bone metabolism. It not only participates in the construction of the osteocyte network but also regulates osteocyte activity, osteoblast differentiation, and osteogenic activities. Meanwhile, in vascular endothelial cells, Cx43 plays an important role in angiogenesis and maintaining vascular homeostasis, and is closely related to the vascularization of bone tissue. In addition, Cx43 is associated with the release of prostaglandin E2 (PGE2). GCs can inhibit the activity of Cx43, reduce the release of PGE2, and disrupt the balance of bone metabolism. Studies have shown that measuring changes in the expression level of Cx43 is expected to become an early diagnostic biomarker for GCs-induced ONFH. Enhancing its expression through small - molecule drugs, biological agents, and gene therapy may be potential treatment approaches for ONFH. This article proposes the PI3K/Akt/GSK3β/β-catenin pathway and conducts research on the regulatory mechanism of Cx43-mediated GJ-based intercellular communication, aiming to provide new ideas for the treatment of ONFH and bone metabolism-related diseases.

This study aimed to compare the differentiation potential of dental pulp-derived mesenchymal stem cells (DP-MSCs) and hair follicle-derived mesenchymal stem cells (HF-MSCs), which originated from the ectoderm. Dental pulps were separated from the extracted wisdom teeth during dental surgery, and Hair follicles were extracted from the scalp of patients undergoing hair transplantation. We cultivated the cell in cell culture media, supplemented with additional nutrients. After the fourth passage, the homogeneous population of DP-MSCs and HF-MSCs was analyzed for the surface markers (CD73, CD90, and CD105) by fluorescence-activated cell sorting. In vitro, the multi-lineage differentiation potential for both the MSCs was tested with respective induction media such as osteogenic, chondrogenic, adipogenic, and insulin-producing cells. Following the fourth passage, identical fibroblast-like cells were noted in each culture plate. Mesenchymal stem cell marker was expressed in both DP-MSCs and HF-MSCs. Both the DP-MSCs and HF-MSCs exhibited similar differentiation potential toward osteogenic, chondrogenic, and adipogenic differentiation. However, there was a difference in the differentiation potential into IPCs. HF-MSCs showed higher C-peptide and insulin secretion response to glucose, PDX1, and Insulin gene expression compared to DP-MSCs. These findings suggest that although DP-MSCs and HF-MSCs showed similar stemness properties, they differ in their differentiation potential towards insulin-producing cells (IPCs). This is the first report showing the potential of HF-MSCs to generate IPCs, revealing hair follicles as a novel and promising source for autologous stem cell therapy in diabetes. The generated islet organoids can be used for diabetic drug toxicity testing and screening.

Neurons, distributed throughout the body, regulate various bodily functions. The recovery of the nervous system is often slow and can be irreversible. Currently, the approach of using mesenchymal stem cells (MSCs) in conjunction with conventional treatments for nervous system injuries is being explored. Nanoemulsions are systems designed for the nanoscale delivery of drug cargoes. Foeniculum vulgare (F. vulgare), a medicinal plant long utilized in complementary medicine, is the focus of this study. The aim is to utilize nanoemulsions of fennel to induce the differentiation of MSCs into neural-like cells in vitro.

Human adipose-derived mesenchymal stem cells (hADSCs) were commercially purchased. These cells were cultured in DMEM medium containing 10 % fetal bovine serum and 1 % penicillin-streptomycin antibiotic. Based on a sequential extraction method, n-hexane (Hex), ethyl acetate (EtAc), and ethanolic extracts were obtained from the seeds of F. vulgare. To prepare the F. vulgare extract nanoemulsion, the aqueous phase (distilled water), the oily part (F. vulgare extract), Span 80 and Tween 20 were used. The optimal dose of F. vulgare nanoemulsion was determined using the MTT assay and acridine orange/ethidium bromide (AO/EB) staining. Neural differentiation was induced using a specialized differentiation medium on the MSCs, with the prepared nanoemulsions acting as inducers. The neural differentiation of the human differentiated hADSCs was studied and evaluated through Real-time PCR and immunocytochemistry (ICC) techniques on days 7 and 14.

The results obtained from the MTT and AO/EB tests indicated that the optimal dose of F. vulgare nanoemulsions is 1 μg/ml. Analysis of neural differentiation index gene expression revealed a significant (P ≤ 0.05) upregulation of MAP-2, β-tubulin III, and NSE genes on days 7 and 14 following treatment with the nanoemulsions. It is noteworthy that the nanoemulsion prepared from the hexane extract of the plant showed a significant increase in the expression of marker genes in the process of neural differentiation. Protein expression analysis demonstrated an increase in MAP-2, β-tubulin III, and NSE (gamma enolase) proteins in response to the nanoemulsion inducers compared to the control group (TCPS).

Overall, our findings indicate that F. vulgare nanoemulsions have a positive effect on the expression of genes and proteins related to neural differentiation in hADSCs. The proposed protocol may serve as a potential therapeutic strategy in complementary medicine for patients seeking to improve injuries to the nervous system. However, further studies and performance measurements are necessary in future research to confirm these results.

To investigate the safety and tolerability of subconjunctival injection of three escalating doses of allogeneic bone marrow-derived mesenchymal stromal cells (MSCs) in patients with persistent corneal epithelial defect/disease (PCED).

Prospective single-center open label phase 1b clinical trial.

Patients with PCED in the setting of neurotrophic keratitis and/or limbal stem cell deficiency.

A dose escalation study design was used. The first three patients received a subconjunctival injection of 1 × 106 MSCs/50 μL suspension; subsequently, three participants were treated with 1 subconjunctival injection of 3 × 106 MSCs/150 μL; and two participants received 2 subconjunctival injections of 3 × 106 MSCs/150 μl in 2 conjunctival sites.

The primary outcome was the safety of the treatment determined on day 28 post-injection. Ocular surface toxicity and other ocular or systemic treatment emergent adverse events (TEAEs) were assessed at 1, 7, 14, 28 and 90 days. Demonstration of safety on day 28 was required before escalating to the next higher dose. Changes in the PCED were also monitored.

Eight participants completed the 90-day study. All 3 doses of subconjunctival MSCs were well tolerated. No participant developed ocular surface toxicity or other ocular or systemic TEAEs. The size of the PCED improved in 5 (63 %) patients; it increased in 2 (25 %) patients; and no progressive improvement was observed with dose escalation.

Subconjunctival administration of MSCs was safe and well tolerated with no systemic or ocular toxicity in patients with PCED. Improvement in epithelial defect size was observed in 63 % of the participants.

Idiopathic Pulmonary Fibrosis (IPF) is a progressive interstitial lung disease marked by increasing dyspnea and respiratory failure. The underlying mechanisms remain poorly understood, given the complexity of its pathogenesis. This review investigates the microenvironment of IPF to identify novel mechanisms and therapeutic avenues. Studies have revealed that various cell types, including alveolar epithelial cells, fibroblasts, myofibroblasts, and immune cells, are integral to disease progression, engaging in cellular stress responses and inflammatory regulation via signaling pathways such as TGF-β, Wnt, mTOR, and ROS. Non-coding RNAs, particularly miRNAs, are critical in IPF and may serve as diagnostic and prognostic biomarkers. Regarding treatment, mesenchymal stem cells (MSCs) and their extracellular vesicles (EVs) or non-vesicular derivatives offer promise by modulating immune responses, enhancing tissue repair, and inhibiting fibrosis. Additionally, alterations in the lung microbiota are increasingly recognized as a contributing factor to IPF progression, offering fresh insights into potential treatments. Despite the encouraging results of MSC-based therapies, the precise mechanisms and clinical applications remain subjects of ongoing research. This review emphasizes the significance of the IPF microenvironment and highlights the need for further exploration to develop effective therapies that could enhance patient outcomes.

Fine particulate matter (PM2.5) exposure significantly exacerbates respiratory morbidity, particularly in asthmatic individuals, highlighting an urgent need for effective therapeutic interventions. In this study, we evaluated the therapeutic potential and underlying mechanisms of human dental pulp stem cells (hDPSCs), a promising mesenchymal stem cell population, in mitigating airway inflammation in mice with PM2.5-induced asthma exacerbation.

In a PM2.5-exacerbated ovalbumin (OVA)-asthma murine model, hDPSCs were intravenously administered with MCC950 (NLRP3 inhibitor) as positive control, systematically evaluating their therapeutic effects on airway inflammation and pyroptosis through pulmonary function tests, histopathological examination, cytological and molecular analyses.

The administration of hDPSCs ameliorated airway inflammation. Moreover, hDPSCs further alleviated Th2 inflammation and decreased serum IgE concentrations, along with a decrease in eosinophils in BALF. At the same time, interleukin-1β (IL-1β) and IL-18 levels in BALF and caspase-1 activity in lung tissues were reduced. In addition, immunohistochemistry showed that the expression levels of NLRP3, caspase-1, GSDMD, cleaved capsase-1 and IL-1β were reduced. The western blot results also showed that the expression level of NLRP3/caspase-1/GSDMD/cleaved capsase-1 in the classical pathway of pyroptosis decreased after hDPSCs intervention.

These findings provided the first evidence that hDPSCs transplantation attenuated allergic airway inflammation and mucus secretion in mice with PM2.5-induced asthma exacerbation. Thus, hDPSCs exert these protective effects through suppression of the NLRP3/caspase-1/GSDMD-mediated pyroptosis pathway, suggesting their potential as a novel cell-based therapy for PM2.5 inhalation-mediated asthma.

Understanding and exploiting bioelectric signaling pathways and physicochemical properties of materials that interface with living tissues is central to advancing tissue regeneration. In particular, the emerging field of bioelectronics leverages these principles to develop personalized, minimally invasive therapeutic strategies tailored to the dynamic demands of individual patients. By integrating sensing and actuation modules into flexible, biocompatible devices, clinicians can continuously monitor and modulate local electrical microenvironments, thereby guiding regenerative processes without extensive surgical interventions. This review provides a critical examination of how fundamental bioelectric cues and physicochemical considerations drive the design and engineering of next-generation bioelectronic platforms. These platforms not only promote the formation and maturation of new tissues across neural, cardiac, musculoskeletal, skin, and gastrointestinal systems but also precisely align therapies with the unique structural, functional, and electrophysiological characteristics of each tissue type. Collectively, these insights and innovations represent a convergence of biology, electronics, and materials science that holds tremendous promise for enhancing the efficacy, specificity, and long-term stability of regenerative treatments, ushering in a new era of advanced tissue engineering and patient-centered regenerative medicine.

We explore the interaction between corneal immunity and mesenchymal stem/stromal cells (MSCs) and their potential in treating corneal and ocular surface disorders. We outline the cornea's immune privilege mechanisms and the immunomodulatory substances involved. In this realm, MSCs are characterized by their immunomodulatory properties and regenerative potential, making them promising for therapeutic application. Therefore, we focus on the role of MSCs in immune-mediated corneal diseases such as dry eye disease, corneal transplantation rejection, limbal stem cell deficiency, and ocular graft-versus-host disease. Preclinical and clinical studies demonstrate MSCs' efficacy in promoting corneal healing and reducing inflammation in these conditions. Overall, we emphasize the potential of MSCs as innovative therapies in ophthalmology, offering promising solutions for managing various ocular surface pathologies.

Maxillofacial neurological disorders include a range of disorders affecting the cranial nerves, which can be caused by a variety of reasons, including infection, trauma, tumor, and surgical complications, resulting in severe dysfunction, and the study of new approaches for the treatment of these disorders is crucial for the restoration of sensory and motor functions of the face. In recent years, due to the excellent tissue regenerative ability of mesenchymal stromal cells (MSCs), research on MSCs and MSC-derived exosomes has been progressively deepened, bringing many new perspectives to the therapeutic strategies for many diseases. Facial nerve regeneration is a complex process involving various pathophysiological mechanisms and therapeutic strategies to restore nerve function after injury. And the rapid development of stem cell tissue engineering has greatly facilitated the research process of facial nerve regeneration. In this paper, we review the characteristics of MSCs and neural stem cells (NSCs), the roles they play in the neural microenvironment and the mechanisms that promote nerve regeneration, summarize the research progress of MSCs in the treatment of maxillofacial neurological disorders, and highlight the promising directions for future development.

The severity and threat posed by inflammation are well documented, and lipopolysaccharides (LPS), as important inducers of inflammatory responses, are widely recognized for studying host immunity and the resulting tissue and organ damage. The LPS-induced disease model, triggers a remarkable release of inflammatory factors, immune and coagulation dysfunction, and damage to vital organs such as the brain, lungs, heart, liver, and kidneys. Recently, the role of mesenchymal stem cells (MSCs) in various clinical diseases has garnered significant attention due to their immunomodulatory, anti-inflammatory, tissue healing, anti-apoptotic, and antibacterial properties. Despite the common use of LPS models to induce disease models and simulate acute inflammation, the integration of stem cell therapy within these models remains underexplored. This article integrates the LPS induced animal model and reviews the current evidence regarding the therapeutic mechanisms of stem cells in LPS-induced disease models across various human body systems. Furthermore, this review predicts and hypothesizes the feasibility and potential of using stem cells in disease models that have not yet been extensively studied, based on existing animal inflammation models.

Mesenchymal stem cells (MSCs) have several important properties that make them desirable for regenerative medicine. These properties include immunomodulatory ability, growth factor production, and differentiation into various cell types. Despite extensive research and promising results in clinical trials, our understanding of MSC biology, their mechanism of action, and their targeted and routine use in clinics is limited. Differentiation of human MSCs (hMSCs) is a complex process influenced by various elements such as growth factors, pharmaceutical compounds, microRNAs, 3D scaffolds, and mechanical and electrical stimulation. Research has shown that different culture conditions can affect the differentiation potential of hMSCs obtained from multiple fetal and adult sources. Additionally, it seems that what affects the differentiation capacities of these cells is their secretory characteristics, which are influenced by the origin of the cells and the local microenvironment where the cells are located. The review can provide insights into the microenvironment-based mechanisms involved in MSC differentiation, which can be valuable for future therapeutic applications.

Diabetic keratopathy is a prevalent but sometimes ignored visual condition in diabetic patients, which significantly affects patients with diabetes mellitus (DM) in terms of their visual acuity. Exosomes regulate diabetes-related conditions like diabetic keratopathy (DK) by secreting their components into the body.

Aim to investigate the effect and mechanism of mesenchymal stem cell (MSC)-derived exosome miR-125a-5p on DK.

Transmission electron microscopy, along with nanoparticle tracking analysis, was used to determine the morphology and size of exosomes. To evaluate cell viability, proliferation, and migration, Western blotting and RT-qPCR methods were used. CCK-8, cell cloning, and scratch assays were used to measure protein levels and mRNA expression.

High glucose treatment of corneal epithelial cells weakened cell viability, proliferation and migration, and the level of miR-125a-5p was significantly reduced. It has been proposed that elevated levels of miR-125a-5p could enhance cell viability, proliferation, and migration, can inhibit endoplasmic reticulum stress induced by high glucose, which is the same as the effect of endoplasmic reticulum stress inhibitors.

Mouse bone marrow MSC-derived exosome miR-125a-5p repairs corneal epithelial cell viability and proliferation as well as migration ability to improve DK by inhibiting high glucose-induced endoplasmic reticulum stress.

Endochondral ossification generates most of the load-bearing bones, recapitulating it in human cells remains a challenge. Here, we report generation of SOX9+ sclerotomal progenitors (scl-progenitors), a mesenchymal precursor at the pre-condensation stage, from human pluripotent stem cells and development of osteochondral induction methods for these cells. Upon lineage-specific induction, SOX9+ scl-progenitors have not only generated articular cartilage but have also undergone spontaneous condensation, cartilaginous anlagen formation, chondrocyte hypertrophy, vascular invasion, and finally bone formation with stroma, thereby recapitulating key stages during endochondral ossification. Moreover, self-organized growth plate-like structures have also been induced using SOX9+ scl-progenitor-derived fusion constructs with chondro- and osteo-spheroids, exhibiting molecular and cellular similarities to the primary growth plates. Furthermore, we have identified ITGA9 as a specific surface marker for reporter-independent isolation of SOX9+ scl-progenitors and established a culture system to support their expansion. Our work highlights SOX9+ scl-progenitors as a promising tool for modeling human skeletal development and bone/cartilage bioengineering.

The antidiabetic drug metformin possesses antioxidant and cell protective effects including in neuronal cells, suggesting its potential use for treating neurodegenerative diseases. This study aimed to assess metformin's effects on viability and antioxidant activity in human-induced pluripotent stem cell (hiPSC)-derived neurons under varying concentrations and stress conditions. Six lines of hiPSC-derived neuronal progenitors derived from healthy human iPSCs were treated with metformin (1-500 µM) on day 18 of differentiation. For mature neurons (day 30), three concentrations (10 µM, 50 µM, and 100 µM) were used to assess cytotoxicity. MG132 proteasomal inhibitor and sodium arsenite (NaArs) were used to investigate oxidative stress, and 50 µM of metformin was tested for its protective effects against oxidative stress in hiPSC-derived neurons. Metformin treatment did not affect cell viability, neuronal differentiation, or trigger reactive oxygen species (ROS) generation in healthy hiPSC-derived motor neurons. Additionally, mitochondrial membrane potential (MMP) loss was not observed at 50 µM metformin. Metformin effectively protected neurons from stress agents and elevated the expression of antioxidant genes when treated with MG132. However, an interplay between MG132 and metformin resulted in lower expression of Nrf2 and NQO1 compared to the MG132 group alone, indicating reduced JC-1 aggregate levels due to MG132 proteasomal inhibition. Metformin upregulated antioxidant genes in hiPSC-derived neurons under stress conditions and protected the cells from oxidative damage.

Hip osteoarthritis constitutes a prevalent condition among individuals aged 55 and above, serving as one of the primary triggers for joint discomfort and impairment, and marking a substantial origin of chronic pain particularly affecting the elderly population. Our article provides an exhaustive summary of the mechanisms of action, therapeutic efficacy, and potential adverse consequences associated with novel therapeutic modalities including glucocorticoids, hyaluronic acid, platelet-rich plasma, mesenchymal stem cells, and stromal vascular fraction. Concurrently, we conducted a comprehensive evaluation of the clinical efficacy and potential applications of various medications.

In comparison to physical therapy, oral analgesics, and other nonsurgical modalities, intra-articular injection therapy is characterized by enhanced safety and greater efficacy. Moreover, when contrasted with surgical intervention, intra-articular injection demonstrates a lower degree of invasiveness and incurs fewer adverse reactions. Intra-articular treatments have shown excellent local efficacy while significantly minimizing adverse reactions in patients. These methods hold significant potential for development but require comprehensive research and thorough discussion within the academic community.

The regenerative capacity of hair follicles is fundamentally influenced by the intricate interactions between hair follicle stem cells (HFSCs) and their microenvironment. Our study presents a novel strategy for hair regeneration, highlighting the synergistic relationship between dermal papilla cell-derived exosomes (DPC-Exos) and collagenous sequences of Human a1(XVII) Chain (CS-COL17A1) in modulating HFSC activity via the hsa-novel-238a-CASP9 axis. We characterized DPC-Exos using nanoparticle tracking analysis and transmission electron microscopy and confirmed, their purity with the exosomal markers CD81, CD63, and CD9.A dose-dependent CCK-8 assay showed that both DPC-Exos and CS-COL17A1 significantly improved HFSC viability. Scratch and Transwell assays showed improved HFSC migration after treatment. MiRNA sequencing revealed a significant upregulation of hsa-novel-238a in HFSCs after treatment with DPC-Exos and CS-COL17A1, suggesting its involvement in the regulation of HFSCs activity. A dual-luciferase assay confirmed that hsa-novel-238a directly targets the CASP9 gene, elucidating the underlying molecular mechanisms. The combined application of DPC-Exos and CS-COL17A1 significantly improved HFSC migration and proliferation (p < 0.01), highlighting the importance of the hsa-novel-238a-CASP9 axis. This research provides insights into the regulatory network of exosomes and CS-COL17A1, paving the way for innovative therapeutic approaches to treat hair loss and enhance hair follicle regeneration through modulation of the hsa-novel-238a-CASP9 axis.

This study seeks to confirm the therapeutic effectiveness of TRFD in inhibiting adipogenesis and promoting osteogenesis in primary osteoporosis through the MAPK/HIF-1α signaling pathway. C57BL/6J mice underwent ovariectomy (OVX) to induce osteoporosis. Mice were administered TRFD (Low and high doses)estradiol for a duration of 12 weeks. Bone microarchitecture evaluated using Micro-CT, while serum biomarkers and protein expressions were analyzed through enzyme-linked immunosorbent assay, Western blotting, and immunohistochemistry. Furthermore, BMSC were isolated to show differentiation, Osteogenic and adipogenic induction were performed, including ALP activity and Oil Red O staining. Bioinformatics analysis of RNA sequencing data was conducted to identify differentially expressed genes.

Total flavonoids of Rhizoma Drynariae treatment significantly improved bone microarchitecture and reversed histopathological damage in OVX mice. It increased serum levels of osteogenesis markers (RUNX2, BMP-2) and enhanced MAPK and HIF-1α signaling pathways, The results also showed a significant dose, TFDR enhanced the osteogenic differentiation of BMSCs while suppressing adipogenic differentiation, as demonstrated by increased ALP activity and mineralization, alongside, the expression of lipid markers (PPAR-γ, C/EBPα) was inhibited. Furthermore, MAPK/HIF-1α pathway was confirmed be crucial in mediating these effects.

TRFD exhibits significant therapeutic potential in treating primary osteoporosis by promoting osteogenesis and inhibiting adipogenesis through the MAPK/HIF-1α pathway. These establish an investigation of TRFD as a natural treatment option for managing osteoporosis.

Not applicable.

The use of mesenchymal stem cells (MSCs) from perinatal tissue sources has gained attention due to their availability and lack of significant ethical or moral concerns. These cells have a higher proliferative capability than adult MSCs and less immunogenic or tumorigenesis risk than fetal and embryonic stem cells. Additionally, they do not require invasive isolation methods like fetal and adult MSCs. We reviewed the main biological and therapeutic aspects of perinatal MSCs in a three-part article. In the first part, we revised the main biological features and characteristics of MSCs and the advantages of perinatal MSCs over other types of SCs. In the second part, we provided a detailed molecular background for the main biomarkers that can be used to identify MSCs. In the final part, we appraised the therapeutic application of perinatal MSCs in four major degenerative disorders: degenerative disc disease, retinal degenerative diseases, ischemic heart disease, and neurodegenerative diseases. In conclusion, there is no single specific molecular marker to identify MSCs. We recommend using at least two positive markers of stemness (CD29, CD73, CD90, or CD105) and two negative markers (CD34, CD45, or CD14) to exclude the hematopoietic origin. Moreover, utilizing perinatal MSCs for managing degenerative diseases presents a promising therapeutic approach. This review emphasizes the significance of employing more specialized progenitor cells that originated from the perinatal MSCs. The review provides scientific evidence from the literature that applying these progenitor cells in therapeutic procedures provides a greater regenerative capacity than the original primitive MSCs. Finally, this review provides a valuable reference for researchers exploring perinatal MSCs and their therapeutic applications.

Chronic kidney disease (CKD) has been a growing public medical concern in recent years which calls for effective interventions. Mesenchymal stem cells (MSCs) have garnered increased interest in past decades due to their potential to repair and regenerate damaged tissues. Many clinical trials have highlighted the safety and effectiveness of kidney disease with this novel cell therapy. MSC infusion can improve renal function indices such as glomerular filtration rate, urine protein, serum creatinine, and blood urea nitrogen, while inhibiting immune response by increasing regulatory T cells. The therapeutic mechanisms may be primarily attributed to a function combined with immunomodulation, anti-inflammation, anti-fibrosis, promoting angiogenesis, anti-oxidation, anti-apoptosis, or tissue healing produced by cell secretsome. However, CKD is a broad concept due to many pathological etiologies including diabetes, hypertension, heart disease, immunological damage, a family history of renal failure, and so on. Furthermore, the therapeutic efficacy of MSCs may be influenced by different cell sources, injection methods, medication dosage, or homing proportion. As a result, it is timely and essential to access recent advancements in the MSC application on CKD.

Articular cartilage (AC) is a specialized connective tissue with unique biological and mechanical properties, which depends on the biological effects of each resident chondrocyte and its surrounding extracellular matrix (ECM) to form a unit that operates in a constant and balanced feedback loop. The surface membrane receptors of chondrocytes play a crucial role in the feedback balance of this biological unit. Various biological signals outside chondrocytes, such as water-soluble chemical signal molecules and mechanical signals, are unable to directly enter the cell and must first bind to the plasma membrane receptors to induce changes in the level and activity of intracellular signal transduction molecules. These changes then transmit through signaling cascade pathways into the nucleus, changing the cell phenotype, and producing physiological or pathological changes. Specific chemical and mechanical signals break the feedback balance of cartilage tissue units through membrane receptors. In the ECM environment, the molecular actions of chondrocyte membrane receptors in response to these specific signals, along with associated ion channel receptors, collectively regulate the biological effects of chondrocytes. This leads to decreased chondrocyte survival and an imbalance in ECM regulation, ultimately disrupting the tissue's molecular framework and physiological feedback mechanisms, and resulting in pathological changes in cartilage tissue. To provide insights into addressing the complexities associated with cartilage tissue injury and repair engineering, this review provides a comprehensive overview of the molecular mechanisms and biological implications of chondrocyte membrane receptor-mediated signal transduction, including G protein-coupled receptors (GPCRs), enzyme-linked receptors (tyrosine kinase receptors (TKRs)), and integrin receptors.

Improved in vitro models are needed for regenerative therapy and drug screening. Here, we report on functionally aligned nanoparticle-trapped nanopattern arrays for spatially controlled, precise mesenchymal stem cell differentiation on a single substrate. The arrays comprise nanohole and nanoline arrays fabricated through interference lithography and selectively capture of UiO-67 metal-organic frameworks on nanoline arrays with a 99.8% efficiency using an optimised asymmetric spin-coating method. The UiO-67 metal-organic frameworks contain three osteogenic differentiation factors for sustained release over four weeks. The combination of differentiation factors and patterned array allows for generation of adipocytes, osteoblasts, and adipocyte-osteoblast mixtures on nanohole arrays, nanoline arrays, and at the nanohole-nanoline interface, respectively, with mature osteoblasts exhibiting higher marker expression and mineralisation. The sustained release patterned array holds potential for constructing advanced therapeutic and disease state in vitro cellular models.

Multiple sclerosis (MS), a chronic autoimmune disorder of the central nervous system (CNS), is characterized by inflammation, demyelination, and neurodegeneration, leading to diverse clinical manifestations such as fatigue, sensory impairment, and cognitive dysfunction. Current pharmacological treatments primarily target immune modulation but fail to arrest disease progression or entirely reverse CNS damage. Mesenchymal stem cell (MSC) therapy offers a promising alternative, leveraging its immunomodulatory, neuroprotective, and regenerative capabilities. This review provides an in-depth analysis of MSC mechanisms of action, including immune system regulation, promotion of remyelination, and neuroregeneration. It examines preclinical studies and clinical trials evaluating the efficacy, safety, and limitations of MSC therapy in various MS phenotypes. Special attention is given to challenges such as delivery routes, dosing regimens, and integrating MSCs with conventional therapies. By highlighting advancements and ongoing challenges, this review underscores the potential of MSCs to revolutionize MS treatment, paving the way for personalized and combinatory therapeutic approaches.

To test the efficacy of nanocarrier (NC) mediated mesenchymal stem cell (MSC) therapy for liver regeneration following thermal ablation of porcine livers.

Liver radiofrequency ablation was performed in 18 swines divided into MSC, MSC + NC and control groups. The test groups received infusion of MSC or MSC + NC labeled with enhanced green fluorescent protein (eGFP) via hepatic artery. MSC + NC group had MSCs coated with dendrimer nanocarrier complexed with I-Domain of lymphocyte function-associated antigen-1 (LFA-1). Nanocarriers direct homing of MSCs by binding to its counterpart protein, intercellular adhesion molecule-1 (ICAM-1), which is overexpressed at the periablation margins from inflammation. Ablation cavity reduction by CT volumetry was used as surrogate marker for liver regeneration. Cell proliferation was assessed with Ki67 and HepPar-1 stains. GFP identified MSC derived cells.

Total number of ablations in control animals were 13 across 4 animals. In the MSC group, there were 23 ablations across 6 animals, and in MSC + NC group there were 21 ablations across 6 animals. Ablation cavity volume reduction from day 0 to 30 were 64.4 ± 15.0%, 61.5 ± 12.9% and 80.3 ± 9.4% for control, MSC and MSC + NC groups, respectively (MSC + NC vs MSC: p < 0.001, MSC + NC vs. control: p = 0.001). GFP+ cell count at margins was 426.8 ± 193.2 for MSC group and 498.6 ± 235.2 for MSC + NC group (p = 0.01). The mean Ki67 and HepPar-1 staining at margins were 9.81 ± 4.5% and 6.12 ± 4.2% for MSC + NC group versus 7.59 ± 3.7% and 5.09 ± 3.7% for MSC group, respectively (P < 0.001 and P = 0.09, respectively).

Nanocarrier-mediated MSC therapy promotes liver regeneration by engrafting MSCs at ablation margins, potentially making liver-directed therapy viable for patients with severe liver dysfunction. This technology may also benefit other solid organs.

Paracrine factors secreted by mesenchymal stem/stromal cells (MSCs) have been demonstrated to have significant therapeutic potential. The secretome profiles of MSCs variate depending on culture conditions. Generally, the effects of a single preconditioning strategy on secretome profiles of MSCs were investigated. However, until now, there has been no study examining the combinatory effects of different preconditioning strategies in a comparative manner. This study aimed to evaluate the secretome contents of conditioned media obtained from human umbilical cord-derived MSCs cultured in 2- or 3-dimensional (D) culture systems preconditioned with deferoxamine (DFS) or dimethyloxalylglycine (DMOG). Immunocytochemical analysis showed that MSCs preconditioned with DFS or DMOG have increased nuclear hypoxia-inducible factor-1α expression. Transmission electron microscopic analysis showed that cells preconditioned with DFS or DMOG have increased autophagic vesicles, which could be attributed to altered energy metabolism under hypoxic conditions. It was revealed that hypoxia-mimetic agents added to the 2D-, or 3D-culture environment raised total protein concentrations per cell along with vascular endothelial growth factor. The concentrations of glial cell-derived neurotrophic factor (GDNF) and nerve growth factor (NGF) were differentially regulated in 2D-, and 3D-culture system, that the secretions of GDNF and NGF per cell were more prominent in 3D- and 2D-culture systems, respectively. These findings indicate that hypoxic conditions alone significantly elevate total protein concentrations, while the contribution of the 3D environment is more modest than initially anticipated. However, concentrations of secreted growth factors may be affected differently depending on the topography of the culture condition and the types of hypoxia mimetic agents.

The regenerative potential of mesenchymal stromal/stem cells (MSCs) has been extensively studied in clinical trials in the past decade. However, despite the promising regenerative properties documented in preclinical studies, for instance in osteoarthritis (OA), the therapeutic translation of these results in patients has not been fully conclusive. One factor contributing to this therapeutic barrier could be the presence of senescent cells in OA joints.

This study evaluated a novel approach to OA treatment by combining adipose tissue-derived MSCs (AD-MSCs) with rapamycin, a clinically approved immunosuppressive drug with anti-senescence properties. First, rapamycin effects on senescence and fibrosis markers were investigated in freshly isolated OA chondrocytes by immunostaining. Next, the in vitro differentiation capacities of AD-MSCs, their regulatory immune functions on activated immune cells and their regenerative effects on OA chondrocyte signature were assessed in the presence of rapamycin.

In OA chondrocytes, rapamycin reduced the senescence marker p15INK4B and the fibrosis marker COL1A1 without affecting the expression of the master chondrogenic markers SOX9 and COL2. Rapamycin also enhanced AD-MSC differentiation into chondrocytes and reduced their differentiation into adipocytes. In addition, rapamycin improved AD-MSC immunoregulatory functions by promoting the expression of immunosuppressive factors, such as IDO1, PTGS2 and also CD274 (encoding PD-L1). Finally, RNA sequencing analysis showed that in the presence of rapamycin, AD-MSCs displayed improved chondroprotective regenerative effects on co-cultured OA chondrocytes.

Our findings suggest that the rapamycin and AD-MSC combination enhances the therapeutic efficacy of these cells in senescence-driven degenerative diseases such as OA, notably by improving their anti-fibrotic and anti-inflammatory properties.

To assess the therapeutic effects of mesenchymal stem cell (MSC)-derived exosome therapy on periodontal regeneration and identify treatment factors associated with enhanced periodontal regeneration in recent preclinical studies.

Searches were conducted in PubMed, Cochrane Library, EMBASE, and Web of Science databases until October 10, 2024. A risk of bias (ROB) assessment was performed using the SYRCLE tool. Osteogenic-related parameters were used as the primary outcome measures.

In total, 1360 articles were identified, of which 17 preclinical studies were based on MSC-derived exosome therapy, and they demonstrated a beneficial effect on BV/TV (SMD = 13.99; 95% Cl = 10.50, 17.48; p < 0.00001), CEJ-ABC (SMD = -0.22; 95% Cl = -0.31, -0.13; p < 0.00001), BMD (SMD = 0.29; 95% Cl = 0.14, 0.45; p = 0.0002), and Tp.Sp (SMD = -0.08; 95% Cl= -0.15, -0.02; p = 0.02) compared with the control group. However, no significant differences were observed in Tp.Th (SMD = 0.03; 95% CI = 0.00, 0.07; p = 0.09) between the exosome-treated group and control group. Additionally, subgroup analysis indicated that preconditioned exosomes (p = 0.03) significantly improved BV/TV. In contrast, there were no significant differences in the enhancement of BV/TV with respect to the application method (p = 0.29), application frequency (p = 0.10), treatment duration (p = 0.15), or source of MSCs (p = 0.31).

MSC-derived exosomes show great promise for enhancing the quality of periodontal regeneration. However, more standardized and robust trials are needed to reduce heterogeneity and bias across studies and to confirm the therapeutic parameters associated with the enhancement of periodontal regeneration by MSC-derived exosomes.

CRD42024546236.

The risk of kidney fibrosis is significantly elevated in individuals with diabetes, chronic nephritis, trauma, and other underlying conditions. Concurrently, human umbilical cord blood-derived mesenchymal stem cells (hUCB-MSCs) and their extracellular vesicles (MSC-Exos) have gained prominence in regenerative medicine. In light of these observations, we are undertaking a meta-analysis to elucidate the influence of hUCB-MSCs and MSC-Exos on kidney fibrosis.

To identify eligible trials, we conducted a comprehensive search of the CNKI, PubMed, Web of Science and Wanfang databases from inception to 24 October 2022. Furthermore, the methodological quality of the included studies was evaluated using the Systematic Review Center for Laboratory Animal Experimentation (SYRCLE) risk-of-bias tool. Besides, the weighted standard mean difference (SMD) with a 95% confidence interval (CI) was calculated using the Review Manager 5.4 software. The Stata (12.0) software was employed to assess the impact of factors on outcome heterogeneity and publication bias in the study. A total of 645 related research studies were retrieved, of which 14 that involved 219 experimental animals were included in the study.

In comparison to the control treatment, treatment with Human UCB MSC and MSC-Exos was observed to significantly enhance renal function in animal models of kidney fibrosis. This was evidenced by a reduction in serum creatinine (Scr) levels (p < 0.00001) and blood urea nitrogen (BUN) levels (p < 0.00001), as well as reduction of CD68+ macrophages (p < 0.00001), TdT-mediated dUTP Nick-End labeling (TUNEL)+ tubular cells(p < 0.00001), α-SMA levels (p = 0.0009) and TGF-β1 (p < 0.00001). P < 0.05 is deemed to indicate a statistically significant difference. Alpha-smooth muscle actin (α-SMA) is a specific protein that is normally expressed in myofibroblasts. The term "CD68+ macrophages" refers to macrophages that express the CD68 protein on their cell surface. Both macrophages and myofibroblasts have been linked to the development of kidney fibrosis. In this study, the quantity of CD68+ macrophages and α-SMA was employed as a means of gauging the extent of renal fibrosis. Additionally, transforming growth factor beta 1 (TGF-β1) is a significant cytokine implicated in the pathogenesis of kidney fibrosis. TUNEL-positive tubular cells represent tubular cells undergoing apoptosis. It is hypothesized that this may result in a reduction of tubular apoptosis and a delay in kidney fibrosis, due to the inhibition of the transformation of macrophages into myofibroblasts (MMT) and the disruption of the kidney fibrogenic niche.

The principal findings of this preclinical systematic review indicate that hUCB MSC and MSC-Exos have a substantial protective impact against kidney fibrosis. Kidney transfer remains the final option for traditional renal fibrosis treatment. The lack of donors and high cost make it challenging for many patients to access appropriate treatment. Although this study still suffers from three shortcomings: sample size, methodological consistency and translational challenges, the hUCB MSC and MSC-Exos have been demonstrated to reduce tubular apoptosis and inhibit fibrotic progression. The hUCB MSC and MSC-Exos offer a promising alternative due to their lower price and accessibility. Nevertheless, further high-quality studies are required in the future to address the methodological limitations identified in this review.

Identifier INPLASY2022100104.

Periodontal disease is a highly prevalent disease worldwide that seriously affects people's oral health, including gingivitis and periodontitis. Although the current treatment of periodontal disease can achieve good control of inflammation, it is difficult to regenerate the periodontal supporting tissues to achieve a satisfactory therapeutic effect. In recent years, due to the good tissue regeneration ability, the research on Mesenchymal stromal/stem cells (MSCs) and MSC-derived exosomes has been gradually deepened, especially its ability to interact with the microenvironment of the body in the complex immunoregulatory network, which has led to many new perspectives on the therapeutic strategies for many diseases. This paper systematically reviews the immunomodulatory (including bone immunomodulation) properties of MSCs and their role in the periodontal inflammatory microenvironment, summarizes the pathways and mechanisms by which MSCs and MSC-EVs have promoted periodontal regeneration in recent years, lists potential areas for future research, and describes the issues that should be considered in future basic research and the direction of development of "cell-free therapies" for periodontal regeneration.

Microcarriers for large-scale cell culture have a broader prospect in cell screening compared with the traditional high cost, low efficiency, and cell damaging methods. However, the equal biological affinity to cells has hindered its application. Therefore, based on the antifouling strategy of zwitterionic polymer, we developed a cell-specific microcarrier (CSMC) for shielding non-target cells and capturing mesenchymal stem cells (MSCs), which has characteristics of high biocompatibility, low background noise and high precision. Briefly, [2-(methacryloyloxy) ethyl] dimethyl-(3-sulfopropyl) ammonium hydroxide and glycidyl methacrylate were grafted onto polygalacturonic acid, respectively. The former built a hydration layer through solvation to provide an excellent antifouling surface, while the latter provided active sites for the click reaction with sulfhydryl-modified cell-specific peptides, resulting in rapid immobilization of peptides. This method is applicable to the vast majority of polysaccharide materials. The accurate capture ratio of MSCs by CSMC in a mixed multicellular environment is >95 % and the proliferation rate of MSCs on microcarriers is satisfactory. In summary, this grafting strategy of bioactive components lays a foundation for the application of polysaccharide materials in the biomedical field, and the specific adhesive microcarriers also open up new ideas for the development of stem cell screening as well.

Cerebrovascular accidents, also known as strokes, are the leading cause of permanent disability in society, presenting significant socioeconomic and healthcare costs. They can be caused by ischemic factors or hemorrhages, with ischemic strokes being the most common among the population. Therapies for patients suffering from this condition are limited and primarily focus on acute-phase treatment. In recent years, there has been an increase in cellular therapies, employing Stem Cells to mitigate or eliminate the consequences arising from this disease. Mesenchymal Stem Cells (MSCs) hold substantial therapeutic potential in Nervous System pathologies due to their low antigenicity and capacity to differentiate into various human tissues, such as adipogenic, chondrogenic, and osteogenic tissues. This study conducts a literature review using the "clinical trials" and "Pubmed" database, summarizing all ongoing clinical trials for ischemic strokes that utilize MSCs as treatment.

Endometriosis is characterized as a macrophage-related ailment due to its strong link with immune dysfunction. Understanding the status of macrophage polarization in the context of endometriosis-related infertility is crucial for advancing diagnostic and therapeutic strategies. Our comprehensive review delves into the foundational understanding of macrophages and their profound influence on both endometriosis and infertility. Additionally, we illuminate the complex role of macrophages in infertility and endometriosis specifically. Finally, we focused on four critical dimensions: follicular fluid, the intraperitoneal environment, endometrial receptivity, and strategies for managing endometriosis. It is clear that throughout the progression of endometriosis, the diverse polarization states of macrophages play a pivotal role in the internal reproductive environment of infertile individuals grappling with this condition. Modulating macrophage polarization in the reproductive environment of endometriosis patients could address infertility challenges more effectively, offering a promising pathway for treating infertility associated with endometriosis.

Improvement of cell survival is essential for achieving better clinical outcomes in stem cell therapy. We investigated the effects of hyperoxic pre-treatment (HP) on the viability of human adipose stromal stem cells (ASCs).

MTT and Western blot tests were used to assess cell viability and the expression of apoptosis-related proteins, respectively. For the in-vivo trial, the rats were subjected to renal ischaemia-reperfusion (IR).

The results showed that HP could significantly increase the viability of ASCs and decrease apoptotic markers (Bax/BCL-2 ratio and Caspase-3) compared with control cells. There were some additional effects with regard to the improvement of renal structure and function in the animal model. However, the difference between the treated and non-treated transplanted ASCs failed to reach significance.

These results suggested that HP could increase the survival of ASCs against oxidative stress-induced damages in the in-vitro condition, but this strategy was not highly effective in renal IR.

The transplantation of hematopoietic stem and progenitor cells (HSPCs) or mesenchymal stromal/stem cells (MSCs) for the treatment of a wide variety of diseases has been studied extensively. A challenge with cell-based therapies is that migration to and retention at the target site is often difficult to monitor and quantify. Zirconium-89 (89Zr) is a positron-emitting radionuclide with a half-life of 3.3 days, which allows long-term cell tracking. Para-isothiocyanatobenzyl-desferrioxamine B (Df-Bz-NCS) is the chelating agent of choice for 89Zr-cell surface labelling. We utilised a shortened labelling method, thereby avoiding a 30-60-min incubation step for [89Zr]Zr-Df-Bz-NCS chelation, to radiolabel HSPCs and MSCs with zirconium-89.

Three 89Zr-MSC labelling attempts were performed. High labelling efficiencies (81.30 and 87.30%) and relatively good labelling yields (59.59 and 67.00%) were achieved with the use of a relatively larger number of MSCs (4.425 and 3.855 million, respectively). There was no significant decrease in MSC viability after 89Zr-labeling (p = 0.31). This labelling method was also translatable to prepare 89Zr-HSPC; preliminary data from one preparation indicated high 89Zr-HSPC labelling efficiency (88.20%) and labelling yield (71.06%), as well as good HSPC viability after labelling (68.65%).

Successful 89Zr-MSC and 89Zr-HSPC labelling was achieved, which underlines the prospects for in vivo cell tracking studies with positron emission tomography. In vitro investigations with larger sample sizes and preclinical studies are recommended.

The secretome of primary bovine mammosphere-derived epithelial cells (MDECs) has been shown to exert antimicrobial, regenerative, and immunomodulatory properties in vitro, which warrants its study as a potential biologic treatment with the potential to be translated to human medicine. Currently, the use of the MDEC secretome as a therapy is constrained by the limited life span of primary cell cultures and the decrease of secretome potency over cell passages.

To address these limitations, early-passage bovine MDECs were immortalized using hTERT, a human telomerase reverse transcriptase. The primary and immortal MDECs were compared morphologically, transcriptomically, and phenotypically. The functional properties and proteomic profiles of the secretome of both cell lines were evaluated and compared. All experiments were performed with both low and high passage cell cultures.

We confirmed through in vitro experiments that the secretome of immortalized MDECs, unlike that of primary cells, maintained antimicrobial and pro-migratory properties over passages, while pro-angiogenic effects of the secretome from both primary and immortalized MDECs were lost when the cells reached high passage. The secretome from primary and immortalized MDECs, at low and high passages exerted immunomodulatory effects on neutrophils in vitro.

High passage immortalized MDECs retain a bioactive secretome with antimicrobial, regenerative, and immunomodulatory properties, suggesting they may serve as a consistent cell source for therapeutic use.

Mesenchymal stem cells (MSCs) are widely dispersed in vivo and are isolated from several tissues, including bone marrow, heart, body fluids, skin, and perinatal tissues. Bone marrow MSCs have a multidirectional differentiation potential, which can be induced to differentiate the medium in a specific direction or by adding specific regulatory factors. MSCs repair damaged tissues through lineage differentiation, and the ex vivo transplantation of bone marrow MSCs can heal injured sites. MSCs have different propensities for lineage differentiation and pathological evolution for different diseases, which are crucial in disease progression. In this study, we describe various lineage analysis methods to explore lineage ontology in vitro and in vivo, elucidate the impact of MSC lineage differentiation on diseases, advance our understanding of the role of MSC differentiation in physiological and pathological states, and explore new targets and ideas associated with disease diagnosis and treatment.

For the efficient delivery of a cell therapy a treatment must be provided rapidly, at clinical scale, contain a sufficient active cellular component (biomass), and adhere to a multitude of regulatory requirements. Cryopreservation permits many of these demands to be met more readily. Here we present the cryopreservation and recovery of large volume (2.5L) alginate encapsulated liver cell spheroids (AELS), suitable for use with a novel bioartificial liver device (HepatiCan™) for the treatment of those suffering from acute liver failure (ALF), in regulatory approved cryobags and a cryopreservation process optimised for large volumes. By first assessing the thermal profiles of large scale cryobags with a thermal mimic, the feasibility of cryopreserving a full patient dose simultaneously (3x cryobags containing 833 ml biomass each) was investigated, allowing for small and subsequently large-scale testing of cellular functional recoveries. Work presented here demonstrates that optimised reproducible cooling and warming profiles could be achieved with these large volumes, leading to high biomass recoveries at full clinical scale. The recovered AELS also had high regeneration potential, achieving full pre-freeze viable cell densities within 3 days, indicating that the cell therapy could be delivered rapidly to patients with ALF. This study has presented the feasibility for rapid delivery of large volume cell therapies, whilst further research into improved speed of post-thaw recovery is warranted.