Effect of Postbiotics Derived from Lactobacillus rhamnosus PB01 (DSM 14870) on Sperm Quality: A Prospective In Vitro Study.

Vaginally administered postbiotics derived from Lactobacillus were recently demonstrated to be effective in alleviating bacterial vaginosis and increasing pregnancy rates. However, their potential effect on sperm quality has not been well investigated. This controlled in vitro study aimed to assess the dose- and time-dependent effects of postbiotics derived from Lactobacillus rhamnosus PB01 (DSM 14870) on sperm quality parameters. The experiment was conducted in vitro to eliminate potential confounding factors from the female reproductive tract and vaginal microbiota. Sperm samples from 18 healthy donors were subjected to analysis using Computer-Aided Sperm Analysis (CASA) in various concentrations of postbiotics and control mediums at baseline, 60 min, and 90 min of incubation. Results indicated that lower postbiotic concentration (PB5) did not adversely affect sperm motility, kinematic parameters, sperm DNA fragmentation, and normal morphology at any time. However, concentrations exceeding 15% demonstrated a reduction in progressively motile sperm and a negative correlation with non-progressively motile sperm at all time points. These findings underscore the importance of balancing postbiotic dosage to preserve sperm motility while realizing the postbiotics' vaginal health benefits. Further research is warranted to understand the underlying mechanisms and refine practical applications in reproductive health.

Stable hydrogel adhesion to polydimethylsiloxane enables cyclic mechanical stimulation of 3D-bioprinted smooth muscle constructs.

During normal urination, smooth muscle cells (SMCs) in the lower urinary tract (LUT) are exposed to mechanical signals that have a critical impact on tissue structure and function. Nevertheless, the mechanisms underlying the maintenance of the contractile phenotype of SMCs remain poorly understood. This is due, in part, to a lack of studies that have examined the effects of mechanical loading using three-dimensional (3D) models. In this study, surface modifications of polydimethylsiloxane (PDMS) membrane were evaluated to investigate the effects of cyclic mechanical stimulation on SMC maturation in 3D constructs. Commercially available cell stretching plates were modified with amino or methacrylate groups to promote adhesion of 3D constructs fabricated by bioprinting. After 6 days of stimulation, the effects of mechanical stimulation on the expression of contractile markers at the mRNA and protein levels were analyzed. Methacrylate-modified surfaces supported stable adhesion of the 3D constructs to the membrane and facilitated cyclic mechanical stimulation, which significantly increased the expression of contractile markers at the mRNA and protein levels. These effects were found to be mediated by activation of the p38 MAPK pathway, as inhibition of this pathway abolished the effects of stimulation in a dose-dependent manner. These results provide valuable insights into the role of mechanical signaling in maintaining the contractile phenotype of bladder SMCs, which has important implications for the development of future treatments for LUT diseases.

Correction: Peng et al. Distinct Dominant Lineage from In Vitro Expanded Adipose-Derived Stem Cells (ASCs) Exhibits Enhanced Wound Healing Properties. Cells 2022, 11, 1236.

In the original publication [...].

Tailoring Hydrogel Composition and Stiffness to Control Smooth Muscle Cell Differentiation in Bioprinted Constructs.

BACKGROUND: Reliable in vitro cellular models are needed to study the phenotypic modulation of smooth muscle cells (SMCs) in health and disease. The aim of this study was to optimize gelatin methacrylate (GelMA)/alginate hydrogels for bioprinting three-dimensional (3D) SMC constructs. METHODS: Four different hydrogel groups were prepared by mixing different concentrations (% w/v) of GelMA and alginate: G1 (5/1.5), G2 (5/3), G3 (7.5/1.5), and G4 (7.5/3). GelMA 10% was used as control (G5). A circular structure containing human bladder SMCs was fabricated by using an extrusion-based bioprinter. The effects of the mixing ratios on printability, viability, proliferation, and differentiation of the cells were investigated. RESULTS: Rheological analysis showed that the addition of alginate significantly stabilized the change in mechanical properties with temperature variations. The group with the highest GelMA and alginate concentrations (G4) exhibited the highest viscosity, resulting in better stability of the 3D construct after crosslinking. Compared to other hydrogel compositions, cells in G4 maintained high viability (> 80%), exhibited spindle-shaped morphology, and showed a significantly higher proliferation rate within an 8-day period. More importantly, G4 provided an optimal environment for the induction of a SMC contractile phenotype, as evidenced by significant changes in the expression of marker proteins and morphological parameters. CONCLUSION: Adjusting the composition of GelMA/alginate hydrogels is an effective means of controlling the SMC phenotype. These hydrogels support bioprinting of 3D models to study phenotypic smooth muscle adaptation, with the prospect of using the constructs in the study of therapies for the treatment of urethral strictures.

Sources, Selection, and Microenvironmental Preconditioning of Cells for Urethral Tissue Engineering.

Urethral stricture is a common urinary tract disorder in men that can be caused by iatrogenic causes, trauma, inflammation, or infection and often requires reconstructive surgery. The current therapeutic approach for complex urethral strictures usually involves reconstruction with autologous tissue from the oral mucosa. With the goal of overcoming the lack of sufficient autologous tissue and donor site morbidity, research over the past two decades has focused on cell-based tissue-engineered substitutes. While the main focus has been on autologous cells from the penile tissue, bladder, and oral cavity, stem cells from sources such as adipose tissue and urine are competing candidates for future urethral regeneration due to their ease of collection, high proliferative capacity, maturation potential, and paracrine function. This review addresses the sources, advantages, and limitations of cells for tissue engineering in the urethra and discusses recent approaches to improve cell survival, growth, and differentiation by mimicking the mechanical and biophysical properties of the extracellular environment.

A Comparative Analysis of the Wound Healing-Related Heterogeneity of Adipose-Derived Stem Cells Donors.

Adipose-derived Stem cells (ASCs) are on the verge of being available for large clinical trials in wound healing. However, for developing advanced therapy medicinal products (ATMPs), potency assays mimicking the mode of action are required to control the product consistency of the cells. Thus, greater effort should go into the design of product assays. Therefore, we analyzed three ASC-based ATMPs from three different donors with respect to their surface markers, tri-lineage differentiation, proliferation, colony-forming unit capacity, and effect on fibroblast proliferation and migration, endothelial proliferation, migration, and angiogenesis. Furthermore, the transcriptome of all three cell products was analyzed through RNA-sequencing. Even though all products met the criteria by the International Society for Cell and Gene Therapy and the International Federation for Adipose Therapeutics and Science, we found one product to be consistently superior to others when exploring their potency in the wound healing specific assays. Our results indicate that certain regulatory genes associated with extracellular matrix and angiogenesis could be used as markers of a superior ASC donor from which to use ASCs to treat chronic wounds. Having a panel of assays capable of predicting the potency of the product would ensure the patient receives the most potent product for a specific indication, which is paramount for successful patient treatment and acceptance from the healthcare system.

Potency assays for human adipose-derived stem cells as a medicinal product toward wound healing.

In pre-clinical studies, human adipose-derived stem cells (hASCs) have shown great promise as a treatment modality for healing of cutaneous wounds. The advantages of hASCs are that they are relatively easy to obtain in large numbers from basic liposuctions, they maintain their characteristics after long-term in vitro culture, and they possess low immunogenicity, which enables the use of hASCs from random donors. It has been hypothesized that hASCs exert their wound healing properties by reducing inflammation, inducing angiogenesis, and promoting fibroblast and keratinocyte growth. Due to the inherent variability associated with the donor-dependent nature of ASC-based products, it appears necessary that the quality of the different products is prospectively certified using a set of most relevant potency assays. In this review, we present an overview of the available methodologies to assess the Mode and the Mechanism of Action of hASCs, specifically in the wound healing scenario. In conclusion, we propose a panel of potential potency assays to include in the future production of ASC-based medicinal products.

Distinct Dominant Lineage from In Vitro Expanded Adipose-Derived Stem Cells (ASCs) Exhibits Enhanced Wound Healing Properties.

It has been suggested that immunophenotypically defined lineages within the in vitro expanded adipose-derived stem cell (ASC) may play a beneficial role from the perspective of a personalized intervention. Therefore, to better understand the implications of different surface marker profiles for the functionality, we set out to examine the evolution of ASC-variants based on the co-expression of five bright or eight dim epitopes. At passages P1, P4, and P8, the co-localization of five bright markers (CD73, CD90, CD105, CD166, and CD201), or eight dim markers (CD34, CD36, CD200, CD248, CD271, CD274, CD146, and the Stro-1), was investigated by flow cytometry. Selected subpopulations were isolated using the fluorescence-activated cells sorting from the cryopreserved P4 and analyzed in terms of proliferative and clonogenic properties, trilineage differentiation, and wound healing potential. Only two of the dim epitopes were found in representative subpopulations (SP), and from the P4 onwards, two major combinations featuring the CD274+ (SP1) or the CD274+ CD146+ (SP2) emerged. Upon sorting and growth, both subpopulations assumed new but highly similar clonal profiles, consisting of the CD274+ CD146+ and the CD274+ CD146+ CD248+ phenotypes. The functional analysis revealed that the SP2 surpassed SP1 and the unfractionated cells regarding the growth rate, clonogenic activity, and the wound closure and endothelial tube formation potential. The surface epitopes may be considered a tool to enrich specific functionality and thus improve therapeutic outcomes in dedicated circumstances.

Multiplex Analysis of Adipose-Derived Stem Cell (ASC) Immunophenotype Adaption to In Vitro Expansion.

In order to enhance the therapeutic potential, it is important that sufficient knowledge regarding the dynamic changes of adipose-derived stem cell (ASC) immunophenotypical and biological properties during in vitro growth is available. Consequently, we embarked on a study to follow the evolution of highly defined cell subsets from three unrelated donors in the course of eight passages on tissue culture polystyrene. The co-expression patterns were defined by panels encompassing seven and five cell surface markers, including CD34, CD146, CD166, CD200, CD248, CD271, and CD274 and CD29, CD31, CD36, CD201, and Stro-1, respectively. The analysis was performed using multichromatic flow cytometry. We observed a major paradigm shift, where the CD166-CD34+ combination which was found across all cell subsets early in the culture was replaced by the CD166+ phenotype as the population homogeneity increased with time. At all analysis points, the cultures were dominated by a few major clones that were highly prevalent in most of the donors. The selection process resulted in two predominant clones in the larger panel (CD166+CD34-CD146-CD271- CD274-CD248-CD200- and CD166+CD34+ CD146-CD271-CD274-CD248-CD200-) and one clone in the smaller panel (CD29+CD201+CD36- Stro-1- CD31-). The minor subsets, including CD166+CD34-CD146-CD271+CD274-CD248-CD200- and CD166+CD34+CD146+CD271-CD274-CD248-CD200-, and CD29+CD201-CD36-Stro-1-CD31-, CD29+CD201+CD36-Stro-1+CD31-, and CD29+CD201+CD36+Stro-1-CD31-, in the seven and five marker panels, respectively, were, on the other, hand highly fluctuating and donor-dependent. The results demonstrate that only a limited number of phenotypical repertoires are possible in ASC cultures. Marked differences in their relative occurrence between distinct individuals underscore the need for potency standardization of different ASC preparation to improve the clinical outcome.

Systematic Review of Stem-Cell-Based Therapy of Burn Wounds: Lessons Learned from Animal and Clinical Studies.

Treatment of severe burn wounds presents a daunting medical challenge, and novel approaches promoting healing and reducing scarring are highly desirable. The application of mesenchymal stem/stromal cells (MSCs) has been suggested as a novel treatment. In this paper, we present systematic reviews of pre-clinical and clinical studies of MSC therapy for second- or third-degree thermal burn wounds. Following the Preferred Reporting Items for Systematic Reviews and Meta-Analysis (PRISMA) guidelines, the PubMed and Embase databases were searched, and interventional studies of MSC therapy using rodent models (21 studies) or human burn patients (three studies) were included in the pre-clinical and clinical reviews, respectively, where both overall outcome and wound-healing-phase-specific methodologies and effects were assessed. The pre-clinical studies demonstrated a promising effect of the application of MSCs on several wound healing phases. The clinical studies also suggested that the MSC treatment was beneficial, particularly in the remodeling phase. However, the limited number of studies, their lack of homogeneity in study design, relatively high risk of bias, lack of reporting on mode of action (MOA), and discontinuity of evidence restrict the strength of these findings. This comprehensive review presents an overview of available methodologies to assess the MOA of MSC treatment for distinct wound healing phases. Furthermore, it includes a set of recommendations for the design of high-quality clinical studies that can determine the efficacy of MSCs as a therapy for burn wounds.

Distribution of Stromal Cell Subsets in Cultures from Distinct Ocular Surface Compartments.

PURPOSE: To reveal the phenotypic differences between human ocular surface stromal cells (hOSSCs) cultured from the corneal, limbal, and scleral compartments. METHODS: A comparative analysis of cultured hOSSCs derived from four unrelated donors was conducted by multichromatic flow cytometry for six distinct CD antigens, including the CD73, CD90, CD105, CD166, CD146, and CD34. RESULTS: The hOSSCs, as well as the reference cells, displayed phenotypical profiles that were similar in high expression of the hallmark mesenchymal stem cell markers CD73, CD90, and CD105, and also the cancer stem cell marker CD166. Notably, there was considerable variation regarding the expression of CD34, where the highest levels were found in the corneal and scleral compartments. The multi-differentiation potential marker CD146 was also expressed highly variably, ranging from 9% to 89%, but the limbal stromal and endometrial mesenchymal stem cells significantly surpassed their counterparts within the ocular and reference groups, respectively. The use of six markers enabled investigation of 64 possible variants, however, just four variants accounted for almost 90% of all hOSSCs, with the co-expression of CD73, CD90, CD105, and CD166 and a combination of CD146 and CD34. The limbal compartment appeared unique in that it displayed greatest immunophenotype diversity and harbored the highest proportion of the CD146+CD34- pericyte-like forms, but, interestingly, the pericyte-like cells were also found in the avascular cornea. CONCLUSION: Our findings confirm that the hOSSCs exhibit an immunophenotype consistent with that of MSCs, further highlight the phenotypical heterogeneity in stroma from distinct ocular surface compartments, and finally underscore the uniqueness of the limbal region.

Evolution of ASC Immunophenotypical Subsets During Expansion In Vitro.

Adipose-derived stromal/stem cells (ASCs) are currently being considered for clinical use for a number of indications. In order to develop standardized clinical protocols, it is paramount to have a full characterization of the stem cell preparations. The surface marker expression of ASCs has previously been characterized in multiple studies. However, most of these studies have provided a cross-sectional description of ASCs in either earlier or later passages. In this study, we evaluate the dynamic changes of 15 different surface molecules during culture. Using multichromatic flow cytometry, ASCs from three different donors each in passages 1, 2, 4, 6, and 8 were analyzed for their co-expression of markers associated with mesenchymal stem cells, wound healing, immune regulation, ASC markers, and differentiation capacity, respectively. We confirmed that at an early stage, ASC displayed a high heterogeneity with a plethora of subpopulations, which by culturing became more homogeneous. After a few passages, virtually all ASCs expressed CD29, CD166 and CD201, in addition to canonical markers CD73, CD90, and CD105. However, even at passage 8, there were several predominant lineages that differed with respect to the expression of CD34, CD200 and CD271. Although the significance of remaining subpopulations still needs to be elucidated, our results underscore the necessity to fully characterize ASCs prior to clinical use.

Fabrication and characterization of extracellular matrix scaffolds obtained from adipose-derived stem cells.

Chronic non-healing wounds are detrimental for the quality of life of the affected individuals and represent a major burden for the health care systems. Adipose-derived stem cells (ASCs) are being investigated for the development of novel treatments of chronic wounds, as they have shown several positive effects on wound healing. While these effects appear to be mediated by the release of soluble factors, it is has also become apparent that the extracellular matrix (ECM) deposited by ASCs is essential in several phases of the wound healing process. In this work, we describe an approach to produce ECM scaffolds derived from ASCs in culture. Upon growth of ASCs into an overconfluent cell layer, a detergent-based cell extraction approach is applied to remove the cellular components. The extraction is followed by an enzymatic treatment to remove the residual DNA. The resultant cell-derived scaffolds are depleted of cellular components, display low DNA remnant, and retain the native fibrillar organization of the ECM. Analysis of the molecular composition of the ECM scaffolds revealed that they are composed of collagens type I and III, and fibronectin. The decellularized scaffolds represent a substrate that supports adhesion and proliferation of primary human fibroblasts and dermal microvascular endothelial cells, indicating their potential as platforms for wound healing studies.

Regeneration of the oesophageal muscle layer from oesophagus acellular matrix scaffold using adipose-derived stem cells.

This study explored the feasibility of constructing a tissue engineered muscle layer in the oesophagus using oesophageal acellular matrix (OAM) scaffolds and human aortic smooth muscle cells (hASMCs) or human adipose-derived stem cells (hASCs). The second objective was to investigate the effect of hypoxic preconditioning of seeding cells on cell viability and migration depth. Our results demonstrated that hASMCs and hASCs could attach and adhere to the decellularized OAM scaffold and survive and proliferate for at least 7 days depending on the growth conditions. This indicates adipose-derived stem cells (ASCs) have the potential to substitute for smooth muscle cells (SMCs) in the construction of tissue engineered oesophageal muscle layers.

Pigmentation Is Associated with Stemness Hierarchy of Progenitor Cells Within Cultured Limbal Epithelial Cells.

Ex vivo cultured human limbal epithelial stem/progenitor cells (hLESCs) are the main source for regenerative therapy of limbal stem cell deficiency (LSCD), which is worldwide one of the major causes of corneal blindness. Despite many stemness-associated markers have been identified within the limbal niche, the phenotype of the earliest hLESCs has not been hitherto identified. We sought to confirm or refute the use of tumor protein p63 (p63) and ATP binding cassette subfamily B member 5 (ABCB5) as surrogate markers for hLESCs early within the limbal differentiation hierarchy. Based on a robust fluorescence-activated cell sorting and subsequent RNA isolation protocol, a comprehensive transcriptomic profile was obtained from four subpopulations of cultured hLESCs. The subpopulations were defined by co-expression of two putative stem/progenitor markers, the p63 and ABCB5, and the corneal differentiation marker cytokeratin 3. A comparative transcriptomic analysis yielded novel data that indicated association between pigmentation and differentiation, with the p63 positive populations being the most pigmented and immature of the progenitors. In contrast, ABCB5, either alone or in co-expression patterns, identified more committed progenitor cells with less pigmentation. In conclusion, p63 is superior to ABCB5 as a marker for stemness. Stem Cells 2018;36:1411-1420.

Hypoxia Enhances Differentiation of Adipose Tissue-Derived Stem Cells toward the Smooth Muscle Phenotype.

Smooth muscle differentiated adipose tissue-derived stem cells are a valuable resource for regeneration of gastrointestinal tissues, such as the gut and sphincters. Hypoxia has been shown to promote adipose tissue-derived stem cells proliferation and maintenance of pluripotency, but the influence of hypoxia on their smooth myogenic differentiation remains unexplored. This study investigated the phenotype and contractility of adipose-derived stem cells differentiated toward the smooth myogenic lineage under hypoxic conditions. Oxygen concentrations of 2%, 5%, 10%, and 20% were used during differentiation of adipose tissue-derived stem cells. Real time reverse transcription polymerase chain reaction and immunofluorescence staining were used to detect the expression of smooth muscle cells-specific markers, including early marker smooth muscle alpha actin, middle markers calponin, caldesmon, and late marker smooth muscle myosin heavy chain. The specific contractile properties of cells were verified with both a single cell contraction assay and a gel contraction assay. Five percent oxygen concentration significantly increased the expression levels of α-smooth muscle actin, calponin, and myosin heavy chain in adipose-derived stem cell cultures after 2 weeks of induction (p < 0.01). Cells differentiated in 5% oxygen conditions showed greater contraction effect (p < 0.01). Hypoxia influences differentiation of smooth muscle cells from adipose stem cells and 5% oxygen was the optimal condition to generate smooth muscle cells that contract from adipose stem cells.

Focal Adhesion Kinase Activation Is Necessary for Stretch-Induced Alignment and Enhanced Differentiation of Myogenic Precursor Cells.

Myogenic precursors sense and dynamically respond to mechanical stimulation through complex integrin-mediated mechanotransduction, in which focal adhesion kinase (FAK) is a fundamental intracellular signaling mediator. When skeletal myoblasts are exposed to uniaxial cyclic tensile strain (UCTS), they display uniform alignment and an enhanced rate of differentiation. In this work, we explored the role of FAK activation by using C2C12 myoblasts that were grown on flexible culture plates and exposed to UCTS during the early differentiation phase. After 24 h, the cells oriented perpendicularly to the direction of strain and exhibited an enhanced differentiation profile. Next, the cells were exposed to a strain field that was either kept in the same direction or rotated 90°, in the presence or not of an FAK phosphorylation inhibitor. On reorientation of the strain field by 90°, the cells reassembled their focal adhesions and actin cytoskeleton to regain the perpendicular position with respect to the engaging stress. After blocking the FAK, however, the cells failed to respond to the reoriented strain field and their differentiation was abrogated. Interestingly, when the strain field remained in the same direction, the FAK inhibitor compromised the differentiation, even though there was no evident change in cell orientation. Our data indicate that during exposure to UCTS, the activation of FAK is necessary for the myoblasts to undergo alignment and enhanced differentiation.

Maintaining RNA Integrity for Transcriptomic Profiling of Ex Vivo Cultured Limbal Epithelial Stem Cells after Fluorescence-Activated Cell Sorting (FACS).

BACKGROUND: Transcriptomic profiling of ex vivo cultured human limbal epithelial stem cells (hLESCs) will foster better understanding of corneal physiology and novel treatment paradigms to limbal stem cell deficiency (LSCD). However, currently such profiling studies are hampered due to difficulties with producing sufficient amounts of intact mRNA for deep RNA sequencing (RNA-seq) from subpopulations sorted on the basis of co-expression of membrane and intracellular antigens by fluorescence-activated cell sorting (FACS). METHODS: To address this problem, we systematically analyzed the critical steps, and found that ethanol fixation together with optimized downstream procedures provided a pipeline that yielded high quality total RNA in amounts to readily support the RNA-seq procedure, while still preserving good discrimination between the individual hLESC immunophenotypes. RESULTS: The average RNA integrity number (RIN) was 7.7 ± 0.4, and the average yield was 4.6 ± 1.7 pg of RNA per cell. The sequencing analysis of the isolated RNA produced high quality data with more than 70% of read pairs mapping uniformly to the reference genome and 80% of bases with a Phred score of at least 30. CONCLUSION: In this study, we developed a reliable FACS-based procedure using ethanol as a fixative that would support accurate isolation of limbal epithelial progenitor subpopulations along with RNA yield and quality sufficient to enable deep transcriptomic profiling.

Implications of Extracellular Matrix Production by Adipose Tissue-Derived Stem Cells for Development of Wound Healing Therapies.

The synthesis and deposition of extracellular matrix (ECM) plays an important role in the healing of acute and chronic wounds. Consequently, the use of ECM as treatment for chronic wounds has been of special interest-both in terms of inducing ECM production by resident cells and applying ex vivo produced ECM. For these purposes, using adipose tissue-derived stem cells (ASCs) could be of use. ASCs are recognized to promote wound healing of otherwise chronic wounds, possibly through the reduction of inflammation, induction of angiogenesis, and promotion of fibroblast and keratinocyte growth. However, little is known regarding the importance of ASC-produced ECM for wound healing. In this review, we describe the importance of ECM for wound healing, and how ECM production by ASCs may be exploited in developing new therapies for the treatment of chronic wounds.

Hypoxia enhances the wound-healing potential of adipose-derived stem cells in a novel human primary keratinocyte-based scratch assay.

Preclinical studies have suggested that paracrine factors from adipose-derived stem cells (ASCs) promote the healing of chronic wounds, and that the exposure of ASCs to hypoxia enhances their wound healing effect. To aid the translation of these findings into clinical use, robust wound models are necessary to explore each aspect of wound healing. The aspect of re-epithelization is often studied in a scratch assay based on transformed keratinocytes. However, there are concerns regarding the validity of this model, since these cell lines differ from normal keratinocytes, both in terms of proliferative capacity and differentiation, and sensitivity to environmental cues. In this study, the main challenge of using primary keratinocytes to examine the effects of ASCs was identified to be their different requirements for calcium in the culture media. We confirmed that a high calcium content led to morphological and cytoskeletal changes in primary keratinocytes, and demonstrated that a low calcium content compromised the growth of ASCs. We found that it is possible to perform the wound healing assay with primary keratinocytes, if the conditioned media from the ASCs is dialyzed to reduce the calcium concentration. Additionally, using this model of re-epithelization, conditioned media from normoxic ASCs was shown to markedly increase the rate of wound closure by primary keratinocytes, and this effect was significantly enhanced with media from the hypoxia-exposed ASCs. These findings, which are in line with the observations from previous in vivo studies, highlight the validity of this modified assay to investigate the wound healing properties of ASCs in vitro.