Transplantation of autologous mesenchymal stromal cells in complete cervical spinal cord injury: a pilot study.

Objective: Spinal cord injury (SCI) is a serious condition that can lead to partial or complete paraplegia or tetraplegia. Currently, there are few therapeutic options for these conditions, which are mainly directed toward the acute phase, such as surgical intervention and high-dose steroid administration. Mesenchymal stromal cells (MSC) have been shown to improve neurological function following spinal cord injury. The aim of the study was to evaluate the safety, feasibility, and potential efficacy of MSC transplantation in patients with cervical traumatic SCI. Methods: We included seven subjects with chronic traumatic SCI (> 1 year) at the cervical level, classified as American Spinal Cord Injury Association impairment scale (AIS) grade A. Subjects received two doses of autologous bone marrow derived MSC, the first by direct injection into the lesion site after hemilaminectomy and the second three months later by intrathecal injection. Neurologic evaluation, spinal magnetic resonance imaging (MRI), urodynamics, and life quality questionnaires were assessed before and after treatment. Results: Cell transplantation was safe without severe or moderate adverse effects, and the procedures were well tolerated. Neurological evaluation revealed discrete improvements in sensitivity below the lesion level, following treatment. Five subjects showed some degree of bilateral sensory improvement for both superficial and deep mechanical stimuli compared to the pretreatment profile. No significant alterations in bladder function were observed during this study. Conclusion: Transplantation of autologous MSC in patients with chronic cervical SCI is a safe and feasible procedure. Further studies are required to confirm the efficacy of this therapeutic approach. Clinical trial registration: https://clinicaltrials.gov/study/NCT02574572, identifier NCT02574572.

Multifunctional vitamin D-incorporated PLGA scaffold with BMP/VEGF-overexpressed tonsil-derived MSC via CRISPR/Cas9 for bone tissue regeneration.

Guiding endogenous regeneration of bone defects using biomaterials and regenerative medicine is considered an optimal strategy. One of the effective therapeutic approaches involves using transgene-expressed stem cells to treat tissue destruction and replace damaged parts. Among the various gene editing techniques for cells, clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) is considered as a promising method owing to the increasing therapeutic potential of cells by targeting specific sites. Herein, a vitamin D-incorporated poly(lactic-co-glycolic acid) (PLGA) scaffold with bone morphogenetic protein 2 (BMP2)/vascular endothelial growth factor (VEGF)-overexpressed tonsil-derived MSCs (ToMSCs) via CRISPR/Cas9 was introduced for bone tissue regeneration. The optimized seeding ratio of engineered ToMSCs on the scaffold demonstrated favorable immunomodulatory function, angiogenesis, and osteogenic activity in vitro. The multifunctional scaffold could potentially support stem cell in vivo and induce the transition from M1 to M2 macrophage with magnesium hydroxide and vitamin D. This study highlights the improved synergistic effect of a vitamin D-incorporated PLGA scaffold and a gene-edited ToMSCs for bone tissue engineering and regenerative medicine.

High-dimensional variable selection accounting for heterogeneity in regression coefficients across multiple data sources.

When analyzing data combined from multiple sources (e.g., hospitals, studies), the heterogeneity across different sources must be accounted for. In this paper, we consider high-dimensional linear regression models for integrative data analysis. We propose a new adaptive clustering penalty (ACP) method to simultaneously select variables and cluster source-specific regression coefficients with sub-homogeneity. We show that the estimator based on the ACP method enjoys a strong oracle property under certain regularity conditions. We also develop an efficient algorithm based on the alternating direction method of multipliers (ADMM) for parameter estimation. We conduct simulation studies to compare the performance of the proposed method to three existing methods (a fused LASSO with adjacent fusion, a pairwise fused LASSO, and a multi-directional shrinkage penalty method). Finally, we apply the proposed method to the multi-center Childhood Adenotonsillectomy Trial to identify sub-homogeneity in the treatment effects across different study sites.

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Urine-derived stem cells serve as a robust platform for generating native or engineered extracellular vesicles.

BACKGROUND: Mesenchymal stromal cell (MSC) therapy holds great potential yet efficacy and safety concerns with cell therapy persist. The beneficial effects of MSCs are often attributed to their secretome that includes extracellular vesicles (EVs). EVs carry biologically active molecules, protected by a lipid bilayer. However, several barriers hinder large-scale MSC EV production. A serum-free culturing approach is preferred for producing clinical-grade MSC-derived EVs but this can affect both yield and purity. Consequently, new strategies have been explored, including genetically engineering MSCs to alter EV compositions to enhance potency, increase circulation time or mediate targeting. However, efficient transfection of MSCs is challenging. Typical sources of MSC include adipose tissue and bone marrow, which both require invasive extraction procedures. Here, we investigate the use of urine-derived stem cells (USCs) as a non-invasive and inexhaustible source of MSCs for EV production. METHODS: We isolated, expanded, and characterized urine-derived stem cells (USCs) harvested from eight healthy donors at three different time points during the day. We evaluated the number of clones per urination, proliferation capacity and conducted flow cytometry to establish expression of surface markers. EVs were produced in chemically defined media and characterized. PEI/DNA transfection was used to genetically engineer USCs using transposon technology. RESULTS: There were no differences between time points for clone number, doubling time or viability. USCs showed immunophenotypic characteristics of MSCs, such as expression of CD73, CD90 and CD105, with no difference at the assessed time points, however, male donors had reduced CD73 + cells. Expanded USCs were incubated without growth factors or serum for 72 h without a loss in viability and EVs were isolated. USCs were transfected with high efficiency and after 10 days of selection, pure engineered cell cultures were established. CONCLUSIONS: Isolation and expansion of MSCs from urine is non-invasive, robust, and without apparent sex-related differences. The sampling time point did not affect any measured markers or USC isolation potential. USCs offer an attractive production platform for EVs, both native and engineered.

MSC-derived exosomes attenuates pulmonary hypertension via inhibiting pulmonary vascular remodeling.

BACKGROUND: Pulmonary hypertension (PH) is a serious cardiopulmonary disease with significant morbidity and mortality. Vascular obstruction leads to a continuous increase in pulmonary vascular resistance, vascular remodeling, and right ventricular hypertrophy and failure, which are the main pathological features of PH. Currently, the treatments for PH are very limited, so new methods are urgently needed. Msenchymal stem cells-derived exosomes have been shown to have significant therapeutic effects in PH, however, the the mechanism still very blurry. Here, we investigated the possible mechanism by which umbilical cord mesenchymal stem cell-derived exosomes (hUC-MSC-EXO) inhibited monocrotaline (MCT)-induced pulmonary vascular remodeling in a rat model of PH by regulating the NF-κB/BMP signaling pathway. Our data revealed that hUC-MSC-EXO could significantly attenuate MCT-induced PH and right ventricular hypertrophy. Moreover, the protein expression level of BMPR2, BMP-4, BMP-9 and ID1 was significantly increased, but NF-κB p65, p-NF-κB-p65 and BMP antagonists Gremlin-1 was increased in vitro and vivo. Collectively, this study revealed that the mechanism of hUC-MSC-EXO attenuates pulmonary hypertension may be related to inhibition of NF-κB signaling to further activation of BMP signaling. The present study provided a promising therapeutic strategy for PH vascular remodeling.

Enhanced interaction between genome-edited mesenchymal stem cells and platelets improves wound healing in mice.

Impaired wound healing poses a significant burden on the healthcare system and patients. Stem cell therapy has demonstrated promising potential in the treatment of wounds. However, its clinical application is hindered by the low efficiency of cell homing. In this study, we successfully integrated P-selectin glycoprotein ligand-1 (PSGL-1) into the genome of human adipose-derived mesenchymal stem cells (ADSCs) using a Cas9-AAV6-based genome editing tool platform. Our findings revealed that PSGL-1 knock-in enhanced the binding of ADSCs to platelets and their adhesion to the injured site. Moreover, the intravenous infusion of PSGL-1 -engineered ADSCs (KI-ADSCs) significantly improved the homing efficiency and residence rate at the site of skin lesions in mice. Mechanistically, PSGL-1 knock-in promotes the release of some therapeutic cytokines by activating the canonical WNT/ß-catenin signaling pathway and accelerates the healing of wounds by promoting angiogenesis, re-epithelialization, and granulation tissue formation at the wound site. This study provides a novel strategy to simultaneously address the problem of poor migration and adhesion of mesenchymal stem cells (MSCs).

The Role of Mesenchymal Stem Cells in Modulating Adaptive Immune Responses in Multiple Sclerosis.

Multiple sclerosis (MS) is a chronic autoimmune disease of the central nervous system, leading to significant disability through neurodegeneration. Despite advances in the understanding of MS pathophysiology, effective treatments remain limited. Mesenchymal stem cells (MSCs) have gained attention as a potential therapeutic option due to their immunomodulatory and regenerative properties. This review examines MS pathogenesis, emphasizing the role of immune cells, particularly T cells, in disease progression, and explores MSCs' therapeutic potential. Although preclinical studies in animal models show MSC efficacy, challenges such as donor variability, culture conditions, migratory capacity, and immunological compatibility hinder widespread clinical adoption. Strategies like genetic modification, optimized delivery methods, and advanced manufacturing are critical to overcoming these obstacles. Further research is needed to validate MSCs' clinical application in MS therapy.

The Effects of the Pneumonia Lung Microenvironment on MSC Function.

BACKGROUND: Despite promise in preclinical models of acute respiratory distress syndrome (ARDS), mesenchymal stem cells (MSC) have failed to translate to therapeutic benefit in clinical trials. The MSC is a live cell medicine and interacts with the patient's disease state. Here, we explored this interaction, seeking to devise strategies to enhance MSC therapeutic function. METHODS: Human bone-marrow-derived MSCs were exposed to lung homogenate from healthy and E. coli-induced ARDS rat models. Apoptosis and functional assays of the MSCs were performed. RESULTS: The ARDS model showed reduced arterial oxygenation, decreased lung compliance and an inflammatory microenvironment compared to controls. MSCs underwent more apoptosis after stimulation by lung homogenate from controls compared to E. coli, which may explain why MSCs persist longer in ARDS subjects after administration. Changes in expression of cell surface markers and cytokines were associated with lung homogenate from different groups. The anti-microbial effects of MSCs did not change with the stimulation. Moreover, the conditioned media from lung-homogenate-stimulated MSCs inhibited T-cell proliferation. CONCLUSIONS: These findings suggest that the ARDS microenvironment plays an important role in the MSC's therapeutic mechanism of action, and changes can inform strategies to modulate MSC-based cell therapy for ARDS.

A novel comparison framework for epidemiological strategies applied to age-based restrictions versus horizontal lockdowns.

During an epidemic, such as the COVID-19 pandemic, policy-makers are faced with the decision of implementing effective, yet socioeconomically costly intervention strategies, such as school and workplace closure, physical distancing, etc. In this study, we propose a rigorous definition of epidemiological strategies. In addition, we develop a scheme for comparing certain epidemiological strategies, with the goal of providing policy-makers with a tool for their systematic comparison. Then, we put the suggested scheme to the test by employing an age-based epidemiological compartment model introduced in Bitsouni et al. (2024), coupled with data from the literature, in order to compare the effectiveness of age-based and horizontal interventions. In general, our findings suggest that these two are comparable, mainly at a low or medium level of intensity.

Comprehensive analysis of secretome and transcriptome stability of Wharton jelly mesenchymal stromal cells during good manufacturing practice-compliant production.

BACKGROUND: Mesenchymal stromal cells (MSCs) hold promise for cell-based therapies due to their ability to stimulate tissue repair and modulate immune responses. Umbilical cord-derived MSCs from Wharton jelly (WJ) offer advantages such as low immunogenicity and potent immune modulatory effects. However, ensuring consistent quality and safety throughout their manufacturing process remains critical. RNA sequencing (RNA-seq) emerges as a crucial tool for assessing genetic stability and expression dynamics in cell-based therapeutic products. METHODS: We examined the secretome and transcriptome of WJ-MSC signatures throughout Good Manufacturing Practice (GMP) production, focusing on the performance of total RNA or Massive Analysis of cDNA Ends (MACE) sequencing. RESULTS: Through extensive transcriptomic analysis, we demonstrated consistent stability of WJ-MSC expression signatures across different manufacturing stages. Notably, MACE-seq showed improved identification of key expression patterns related to senescence and immunomodulation. CONCLUSIONS: These findings highlight the potential of MACE-seq as a quality assessment tool for WJ-MSC-based therapies, ensuring their efficacy and safety in clinical applications. Importantly, MACE-seq demonstrated its value in characterizing WJ-MSC-derived products, offering insights that traditional assays cannot provide.

Therapeutic efficacy and in vivo distribution of human umbilical cord-derived mesenchymal stem cell spheroids transplanted via B-Ultrasound-guided percutaneous portal vein puncture in rhesus monkey models of liver fibrosis.

BACKGROUND: Liver fibrosis can progress to end-stage cirrhosis and liver cancer. Mesenchymal stem cells (MSCs) were considered the most promising therapeutic strategy, but most of the MSCs injected intravenously traditionally are trapped in the lungs, rapidly reducing their survival ability. MSC spheroids cultured in 3D have shown higher tolerance to fluid shear stress and better survival than dissociated MSCs. Simulating the route of orthotopic liver transplantation, transplanting MSC spheroids into the liver via hepatic portal vein may impact superior therapeutic effects. METHODS: In the present study, human umbilical cord-derived MSC spheroids (hUC-MSCsp) were transplanted into rhesus monkey models of liver fibrosis via B-ultrasound-guided percutaneous portal vein puncture with minimized body invasion. The therapeutic effect is evaluated through hematology, ultrasound, and pathology. To study the effect of hUC-MSCsp on gene expression in rhesus monkeys with liver injury, transcriptome sequencing analysis was performed on the livers of rhesus monkeys. The distribution of transplanted hUC-MSCsp was traced with RNA scope technology. RESULTS: We found that hUC-MSCsp significantly restored liver function, including ALT, AST, ALB, GLOB and bilirubin. hUC-MSCsp also significantly reduced liver collagen deposition and inflammatory infiltration, and promote dismission of liver ascites. Subsequently, the therapeutic effects were further validated in TGF-ß1/Smad pathway by global transcription profile. The distribution of transplanted hUC-MSCsp were also tracked, and we found that hUC-MSCsp distributed in the liver in a sphere status at 1 h after transplantation. After 16 days, the hUC-MSCsp were dispersed into dissociated cells that were predominantly distributed in the spleen, and a significant number of dissociated cells were still present in the liver. CONCLUSIONS: This study reveals the distributions of transplanted hUC-MSCsp after liver portal vein transplantation, and provides a novel approach and new insights into the molecular events of potential molecular events underlying the treatment of liver fibrosis with hUC-MSCsp.

MSC-extracellular vesicle microRNAs target host cell-entry receptors in COVID-19: in silico modeling for in vivo validation.

BACKGROUND: Coronavirus disease 2019 (COVID-19) has created a global pandemic with significant morbidity and mortality. SARS-CoV-2 primarily infects the lungs and is associated with various organ complications. Therapeutic approaches to combat COVID-19, including convalescent plasma and vaccination, have been developed. However, the high mutation rate of SARS-CoV-2 and its ability to inhibit host T-cell activity pose challenges for effective treatment. Mesenchymal stem cells (MSCs) and their extracellular vesicles (MSCs-EVs) have shown promise in COVID-19 therapy because of their immunomodulatory and regenerative properties. MicroRNAs (miRNAs) play crucial regulatory roles in various biological processes and can be manipulated for therapeutic purposes. OBJECTIVE: We aimed to investigate the role of lyophilized MSC-EVs and their microRNAs in targeting the receptors involved in SARS-CoV-2 entry into host cells as a strategy to limit infection. In silico microRNA prediction, structural predictions of the microRNA-mRNA duplex, and molecular docking with the Argonaut protein were performed. METHODS: Male Syrian hamsters infected with SARS-CoV-2 were treated with human Wharton's jelly-derived Mesenchymal Stem cell-derived lyophilized exosomes (Bioluga Company)via intraperitoneal injection, and viral shedding was assessed. The potential therapeutic effects of MSCs-EVs were measured via histopathology of lung tissues and PCR for microRNAs. RESULTS: The results revealed strong binding potential between miRNA‒mRNA duplexes and the AGO protein via molecular docking. MSCs-EVs reduced inflammation markers and normalized blood indices via the suppression of viral entry by regulating ACE2 and TMPRSS2 expression. MSCs-EVs alleviated histopathological aberrations. They improved lung histology and reduced collagen fiber deposition in infected lungs. CONCLUSION: We demonstrated that MSCs-EVs are a potential therapeutic option for treating COVID-19 by preventing viral entry into host cells.

Harnessing the potential of mesenchymal stem cells-derived exosomes in degenerative diseases.

Mesenchymal stem cells (MSCs) have gained attention as a promising therapeutic approach in both preclinical and clinical osteoarthritis (OA) settings. Various joint cell types, such as chondrocytes, synovial fibroblasts, osteoblasts, and tenocytes, can produce and release extracellular vesicles (EVs), which subsequently influence the biological activities of recipient cells. Recently, extracellular vesicles derived from mesenchymal stem cells (MSC-EVs) have shown the potential to modulate various physiological and pathological processes through the modulation of cellular differentiation, immune responses, and tissue repair. This review explores the roles and therapeutic potential of MSC-EVs in OA and rheumatoid arthritis, cardiovascular disease, age-related macular degeneration, Alzheimer's disease, and other degenerative diseases. Notably, we provide a comprehensive summary of exosome biogenesis, microRNA composition, mechanisms of intercellular transfer, and their evolving role in the highlight of exosome-based treatments in both preclinical and clinical avenues.

Stability of infraslow correlation structure in time-shifted intracranial EEG signals.

It is increasingly understood that the epilepsies are characterized by network pathology that can span multiple spatial and temporal scales. Recent work indicates that infraslow (<0.2 Hz) envelope correlations may form a basis for distant spatial coupling in the brain. We speculated that infraslow correlation structure may be preserved even with some time lag between signals. To this end, we studied intracranial EEG (icEEG) data collected from 22 medically refractory epilepsy patients. For each patient, we selected hour-long background, awake icEEG epochs before and after antiseizure medication (ASM) taper. For each epoch, we selected 5,000 random electrode contact pairs and estimated magnitude-squared coherence (MSC) below 0.15 Hz of band power time-series in the traditional EEG frequency bands. Using these same contact pairs, we shifted one signal of the pair by random durations in 15-s increments between 0 and 300 s. We aggregated these data across all patients to determine how infraslow MSC varies with duration of lag. We further examined the effect of ASM taper on infraslow correlation structure. We also used surrogate data to empirically characterize MSC estimator and to set optimal parameters for estimation specifically for the study of infraslow activity. Our empirical analysis of the MSC estimator showed that hour-long segments with MSC computed using 3-min windows with 50% overlap was sufficient to capture infraslow envelope correlations while minimizing estimator bias and variance. The mean MSC decreased monotonically with increasing time lag until 105 s of lag, then plateaued between 106 and 300 s. Significantly nonzero infraslow envelope MSC was preserved in all frequency bands until about 1 min of time lag, both pre- and post-ASM taper. We also saw a slight, but significant increase in infraslow MSC post-ASM taper, consistent with prior work. These results provide evidence for the feasibility of examining infraslow activity via its modulation of higher-frequency activity in the absence of DC-coupled recordings. The use of surrogate data also provides a general methodology for benchmarking measures used in network neuroscience studies. Finally, our study points to the clinical relevance of infraslow activity in assessing seizure risk.

Targeting miR-29 mitigates skeletal senescence and bolsters therapeutic potential of mesenchymal stromal cells.

Mesenchymal stromal cell (MSC) senescence is a key factor in skeletal aging, affecting the potential of MSC applications. Identifying targets to prevent MSC and skeletal senescence is crucial. Here, we report increased miR-29 expression in bone tissues of aged mice, osteoporotic patients, and senescent MSCs. Genetic overexpression of miR-29 in Prx1-positive MSCs significantly accelerates skeletal senescence, reducing cortical bone thickness and trabecular bone mass, while increasing femur cross-sectional area, bone marrow adiposity, p53, and senescence-associated secretory phenotype (SASP) levels. Mechanistically, miR-29 promotes senescence by upregulating p53 via targeting Kindlin-2 mRNA. miR-29 knockdown in BMSCs impedes skeletal senescence, enhances bone mass, and accelerates calvarial defect regeneration, also reducing lipopolysaccharide (LPS)-induced organ injuries and mortality. Thus, our findings underscore miR-29 as a promising therapeutic target for senescence-related skeletal diseases and acute inflammation-induced organ damage.

Extracellular adenosine oppositely regulates the purinome machinery in glioblastoma and mesenchymal stem cells.

Glioblastoma (GB) is a lethal brain tumor that rapidly adapts to the dynamic changes of the tumor microenvironment (TME). Mesenchymal stem/stromal cells (MSCs) are one of the stromal components of the TME playing multiple roles in tumor progression. GB progression is prompted by the immunosuppressive microenvironment characterized by high concentrations of the nucleoside adenosine (ADO). ADO acts as a signaling molecule through adenosine receptors (ARs) but also as a genetic and metabolic regulator. Herein, the effects of high extracellular ADO concentrations were investigated in a human glioblastoma cellular model (U343MG) and MSCs. The modulation of the purinome machinery, i.e., the ADO production (CD39, CD73, and adenosine kinase [ADK]), transport (equilibrative nucleoside transporters 1 (ENT1) and 2 (ENT2)), and degradation (adenosine deaminase [ADA]) were investigated in both cell lines to evaluate if ADO could affect its cell management in a positive or negative feed-back loop. Results evidenced a different behavior of GB and MSC cells upon exposure to high extracellular ADO levels: U343MG were less sensitive to the ADO concentration and only a slight increase in ADK and ENT1 was evidenced. Conversely, in MSCs, the high extracellular ADO levels reduced the ADK, ENT1, and ENT2 expression, which further sustained the increase of extracellular ADO. Of note, MSCs primed with the GB-conditioned medium or co-cultured with U343MG cells were not affected by the increase of extracellular ADO. These results evidenced how long exposure to ADO could produce different effects on cancer cells with respect to MSCs, revealing a negative feedback loop that can support the GB immunosuppressive microenvironment. These results improve the knowledge of the ADO role in the maintenance of TME, which should be considered in the development of therapeutic strategies targeting adenosine pathways as well as cell-based strategies using MSCs.

A novel extracellular vesicle paradigm for the treatment of COVID-19 induced acute respiratory distress syndrome (ARDS).

Efficacy of mesenchymal stem cells (MSCs) for treatment of acute respiratory distress syndrome (ARDS) suggests bioactive bone marrow MSC extracellular vesicles (BM-MSC EVs) may be effective. A patient with severe COVID-19 associated ARDS who was presumed to expire was treated with a BM-MSC EV preparation (14 doses over two months) as a rescue treatment for refractory COVID ARDS. Near complete reversal of lung inflammation and fibrosis (per computed tomography), near complete restoration of mobility, hospital discharge (3 months) with resumption of normal activities of daily living (one year) and return to work occurred. No adverse events occurred despite repeated dosing of investigational product, highlighting safety of this potential therapy for ARDS.

Isolation and Characterization of Human Dental Pulp Stem Cells Derived from Dental Pulp of Permanent Teeth.

Dental pulp stem cells (DPSCs) are a promising alternative to the source of mesenchymal stem cells (MSCs), as they are readily available in minimally invasive procedures compared to more invasive methods associated with harvesting other MSCs sources. Despite the encouraging pre-clinical outcomes in animal disease models, culture-expanding procedures are needed to obtain a sufficient number of MSCs required for delivery to the damaged site. However, this contributes to increasing regulatory difficulties in translating stem cells and tissue engineering therapy to clinical use. Moreover, discussions continue as to which isolation method is to be preferred when obtaining DPSCs from extracted molars. This protocol describes a simple explant isolation technique of human dental pulp stem cells from the dental pulp of permanent teeth based upon the plastic adherence of MSCs and subsequent outgrowth of cells out of tissue fragments with high efficacy.

A Nonenzymatic Procedure to Obtain Human Mesenchymal Stromal Cells from the Dermis.

Human mesenchymal stromal cells (MSCs) have gained significant interest as cell-based therapeutics for organ restoration in the field of regenerative medicine. More recently, substantial attention has been directed toward cell-free therapy, achieved through the utilization of soluble factors possessing trophic and immunomodulatory properties present in the MSC secretome. This collection of soluble factors can be found either freely in the secretome or packed within its vesicular fraction, known as extracellular vesicles (EVs). MSCs can be derived from various tissue sources, each involving different extraction methods and yielding varying cell amounts. In this study, we describe a nonenzymatic procedure for a straightforward isolation of MSCs from the fetal dermis and the adult dermis. The results demonstrate the isolation of a cell population with a uniform MSC immunophenotype from the earliest passages (approximately 90% positive for the classical MSC markers CD90, CD105, and CD73, while negative for the hematopoietic markers CD34 and CD45, as well as HLA-DR). Additionally, we describe the procedures for cell expansion, banking, and secretome collection.