---

Background and purpose: Vitro cartilage differentiation of mesenchymal stem cells (MSCs) has been noticed in several investigations. In this regard, almost always molecular differentiation of the cells has been examined, while structural and morphological differentiation of them has been ignored. Therefore, the present study examines the structure and ultrastructure of the cartilage differentiated from murine MSCs compared with that of costal cartilage.
Materials and Methods: 2× 105 MSCs isolated from the bone marrow of NMRI mice were pleted by centrifugation and cultured for 21 days in chondrogenic medium. At the end of cultivation period, occurrence of chondrogenic differentiation was confirmed by reverse transcriptase–polymerase chain reaction (RT-PCR) analysis for some cartilage-specific genes. To compare the structure of differentiated tissue with that of natural cartilage, the cartilage was differentiated from MSCs and the cartilage obtained from the same murine rib was prepared for transmission electron microscopy (TEM).
Results: Structural studies indicated that similar to the costal cartilage, the cartilage produced from differentiation of perichondrium-like layer was formed. According to the microscopic images, in contrast to costal chondrocytes, the differentiated cells had euchromatic nucleus and their cytoplasm contained plenty of the organelles involved in active cell secretion. Furthermore, intercellular matrix in differentiated cartilage had a fibrillar appearance.
Conclusion: Our results indicated that the structure of cartilage produced in micro mass culture system is somewhat different from that of costal cartilage. The cells from differentiated tissue seemed to be more active than those from costal cartilage.

---

Background and purpose: Because of theirmultipotency and ease of purification, amplification and ‎immunomodulatory properties Mesenchymal stem cells (MSC), are anideal stem cell source for cell therapies. ‎This study was done to investigat the effect of Rat MSCs on the survival neutrophils.‎ Materials and methods: MSCs was isolated from rat (6-8 weeks) femoral and tibial bone marrow and ‎cultured in DMEM. Then maturation MSCs were incubated with neutrophils isolated from peripheral blood of ‎rat at 37 ° C for 1 h. Neutrophil survival was measured at 6 and 24 h incubation with of MSCs by flow ‎cytometric analysis using An/PI. Data were analyzed by SPSSsoftware, followed by Tukeytest at a significance ‎level of (P<0.05).‎ Results: The 6-hour incubation of neutrophils with MSCs, significantly reduced the percentage of ‎normal cells and increased apoptosis compared to controls and increased cell necrosis was not significant in ‎the treated group than in control. The 24-hour incubation of neutrophils with MSCs, significantly increased the ‎percentage of healthy cells and apoptosis was reduced compared to the control group, and reduced cell ‎necrosis was not significant in the treated groups than in control (p<0.05).‎ Conclusion: In spite of the clinical importance of MSCs, some biological aspects of them including ‎their self-renewal, proliferation and immune modulatory effects are of great therapeutic potential for cell ‎therapy.‎

---

Background and purpose: Mesenchymal stem cells (MSCs) are the most widely used cell sources for cartilage tissue engineering. In the present study, human stem cells were used as a cell source. Scaffolds play an important role in tissue engineering, therefore, this study aimed at evaluating the ability of fibrin scaffolds in chondrogenic differentiation of adipose-derived mesenchymal stem cells (ADMSCs). Materials and methods: In this study, after preparing fibrin scaffold, isolating mesenchymal stem cells from adipose tissue, and confirming their isolation the cells were cultured into fibrin scaffolds. Seven days after cell culture, their potential of survival and growth were evaluated using MTT assay. Finally, 14 days after the end of the chondrogenic differentiation, gene expression analysis and chondrocyte morphology formation was performed by Real time PCR and histology analysis, respectively. Results: The mesenchymal stem cell viability, proliferation and expression of 4 specific chondrogenic genes significantly increased in a fibrin scaffold compared with those of the control group. Moreover, the formation of chondrocyte cells on fibrin scaffold was confirmed. Conclusion: Fibrin scaffold was found suitable for condrogenic differentiation of adipose-derived mesenchymal stem cells.

---

Cancer stem cells are believed to be responsible for the cancer-initiating step and resistance to chemotherapy drugs. Studies have shown that cancer stem cells are silent and have no metabolic activity. The main reasons behind tumors resistant to therapies are lack of activity of cancer stem cells and division of cancer cells. This cell population, like normal stem cells, is capable of self-renewing and responsible for survival of tumor and its genetic and metabolic differences. Cancer stem cells can undergo chemotherapy during the treatment, but, the incidence of secondary tumor occurs due to unequal division of cancer stem cells and new tumor cells grow that are multi resistant to chemotherapeutic agents. Therefore, identifying and characterizing cancer stem cell will lead to a better understanding of its controlling pathways and developing better diagnostic and therapeutic methodologies in basic and clinical cancer researches. In this review the role of cancer stem cells in development of cancer and their heterogenic properties in gene expression and metabolism of the tumor has discussed. Finally, new therapeutic strategies that are often based on the use of nanocarriers are presented.

---

Background and purpose: Angiogenesis provides proper nutrition and helps to the development and spread of cancer cells. Cancer stem cells are a rare population of tumor cells responsible for initiation, spreading and growth of cancer. Angiogenesis occurs more in cancer stem cells compared with other cancer cells. Ibuprofen, as a member of nonsteroidal anti-inflammatory drugs (NSAIDs) group is used for prevention and treatment of certain cancers. In the present study, we investigated the effect of ibuprofen on angiogenesis in gastric cancer stem cells.

Materials and methods: Gastric cancer stem cells of MKN45 cell line was isolated by spheroid colony formation technique. Gastric cancer stem cells were treated with various concentrations of ibuprofen. The angiogenic properties in gastric cancer stem cells treated with ibuprofen was compared with control cells using two-dimensional angiogenesis.

Results: Angiogenesis reduced in the cancer stem cells treated with ibuprofen compared with the control cancer stem cells.

Conclusion: Ibuprofen decreased angiogenesis in gastric cancer stem cells.

---

Background and purpose: Application of three-dimensional scaffolds with the ability to simulate a three-dimensional in vivo environment has opened new perspective on targeted differentiation and therapeutic use of stem cells. In this study we examined the compatibility of CD93 stem cells with biodegradable pcl- gelatin scaffold.

Materials and methods: In this experimental study, three-dimensional scaffolds made of PCL -gelatin using electrospining synthesis and its molecular structure was tested by SEM electron microscopy. The scaffold surface was disinfected by UV ray. The hematopoietic CD93stem cells of those isolated previously were divided into two groups including normal cultured (plate) and culture on scaffolds (scaffold + cell). The survival and growth of the cells were measured through MTT assay and electron microscopy at 7, 14, and 28 days after culturing.

Results: Electron microscopic analysis on the seventh day showed appropriate adhesion of CD93 cells on scaffold fibers and secretion of extracellular matrix. Survival rate of the cells at 7, 14, and 28 days after culturing were not significantly different between the two groups. But at the same days significant differences were observed in the Scaffold + Cell group (P< 0.05).

Conclusion: This study suggests that PCL nanofiber scaffolds has high compatibility with CD93 stem cells and proximity to this scaffold lead to increased survival and growth of the cells. Further studies on the treatment of tissue damage and scarring by CD93 stem cells using this scaffold can be effective in increasing treatment efficiency.

---

Background and purpose: The unique characteristics and potency of stem cells have attracted interest for their use in cell therapy. However, the use of these cells has some limitations and problems such as ethical issues. The aim of this study was to perform a systematic review on the use of embryonic stem cell for clinical therapy targets and investigating its advantages and limitations.

Materials and methods: Data was collected from electronic databases including PubMed, Science direct, Medline, Clinical trial.gov, SID, etc. The search keywords included pluripotent stem cell, Embryonic stem cell (eSC), and stem cell therapy.

Results: Study of published articles and ongoing studies showed that pluripotency, cell viability and low immunogenicity of eSC are amongst the major reasons for their use.

Conclusion: Recent developments in clinical application of eSC make them a major candidate in using stem cells alongside MSC and iPS. But, further studies are needed due to its importance in many aspects.  

---

Background and purpose: Today, increased rate of demand for insulin is predictable due to increasing cases of diabetics in the world. Therefore, it is necessary to develop economic approaches and increasing the production of insulin for the future and medicinal plants could be regarded as a promising prospect for insulin production.
Materials and methods: The Allium ursinum and Silybum marianum were collected. Each herbarium was identified in School of Pharmacy affiliated with Mazandaran University of Medical Sciences and the extract was used by percolation with 70% ethanol extraction, after the solvent was evaporated by using the rotary. After the successful isolation of mesenchymal stem cells (MSCs), Wharton's jelly was derived and approved. Then, the mesenchymal stem cells were differentiated to pancreatic beta cells with two herbal extracts.
Results: Compared with the control group, there was a significant difference between the levels of insulin in the culture medium obtained from the two plants (P= 0.0001). In addition, via specific dithizone staining, the insulin producing cells (IPCs) were proven.
Conclusion: The extracts of Allium ursinum and Silybum marianum were found capable in inducing differentiation of the mesenchymal stem cells derived from Wharton's jelly into IPCs. Allium ursinum was seen with the highest rate of insulin production, while Silybum marianum had the lowest rate of insulin production, therefore, Allium ursinum could be more effective in treatment of diabetes.
 

---

In the recent decade, liver diseases with high mortality rate have became one of the major health problems worldwide. Liver transplantation is the most effective and the standard treatment for decompensated liver disease, but, shortage of available organs and inaccuracy in modeling of the liver diseases are the most limiting issues in treatment.
Regenerative medicine and cell based therapy strategies have provided promising results through cooperating in natural liver regeneration ability or providing liver function support as a bridge before transplantation. Induction of differentiation in different types of stem cells such as embryonic, induced pluripotent stem cells, mesenchymal and hematopoietic stem cells are the most common methods that provide a functional population of hepatocytes to be used in diseases like cirrhosis, cancer and all types of fatty liver diseases, and significantly restore the normal levels of hepatic factors. In addition, mesenchymal and hematopoietic stem cells improve the symptoms of autoimmune diseases by modulatation of immune responces and reduction of inflammation. Other cell treatment strategies are isolating the patient's own hepatocytes in the lab, in vito correction of defected genes and transplanting them back to the patient that can improve the symptoms of genetic disorders. In this study, we first reviewed the basic concepts related to liver diseases, then highlighted the most recent advances in cell-based therapies and regenerative medicine for the treatment of liver disease, along with tissue engineering and bio-artificial liver devices.

---

Background and purpose: Apoptosis is a protective cellular process that plays an important role in the development and homeostasis of natural tissues as well as disease-causing factors. Current study aimed at determining the effect of aerobic training, Vitamin D, and injection of adipose tissue-derived stem cells (ADSCs) on some apoptotic and anti-apoptotic indices of beta-pancreatic cells of streptozotocin (STZ) induced diabetic rats.
Materials and methods: In an experimental study, 80rats (220-240g, 8 weeks old) were randomly divided into 10 groups: Control, Sham, diabetics, ADSCs, exercise, Vitamin D, ADSCs +exercise, ADSCs +Vitamin D, exercise+ Vitamin D, and ADSCs +exercise+ Vitamin D. The exercise protocol included treadmill running at 60-70% VO₂max for 5 days a week/ 6 weeks. In ADSCs receiver groups, human ADSCs (1.5*10⁶ PBS/5mL) were injected to the tail vein. Diabetes was induced by intraperitoneal injection of streptozotocin (60mg/kg) dissolved in citrate buffer, pH 4.5. Bax and Bcl2 values of beta pancreatic cells were measured following homogenization and centrifugation using ELISA. Data analysis was done applying one way analysis of variance and Tukey test.
Results: Significant differences were seen between the levels of Bax and Bcl2a well as the ratio of Bcl-2/Bax between diabetic group and control group and diabetic group and other groups (P=0.001). But, there were no significant differences between other groups (P=0.001).
Conclusion: It seems that all interventions, especially their combination, can have an increasing effect on Bcl2 values and decrease Bax values and the ratio of beta-pancreatic cells. So, they can be used as a Bax pharmacological method to reduce hyperglycemia-induced apoptosis on beta cells. 

---

Background and purpose: Recent studies have demonstrated the promising effects of mesenchymal stem cells (MSCs) in some neurodegenerative diseases and proved their neuroprotective effects. But, the detailed pathways and the ability of MSCs from various sources has not been fully investigated.
Materials and methods: Here, we isolated MSCs from two sources; adipose tissue and dental pulp, and compared the neuroprotective effects of adipose derived stem cells-conditioned media (ADSCs-CM) and dental pulp derived stem cells-conditioned media (DPSCs-CM) on SH-SY5Y cells exposed to Cobaltous chloride(CoCl2) as a model of hypoxia-induced neural damage. SH-SY5Y cells exposed to CoCl2 were treated with ADSCs-CM and DPSCs-CM and the cell viability, apoptosis, and cellular damage were determined by AlamarBlue ^®assay, propidium iodide (PI) test, and lactate dehydrogenase (LDH) assay, respectively.
Results: According to AlamarBlue® results, both ADSCs-CM and DPSCs-CM showed protective effects on SH-SY5Y cells exposed to CoCl₂ at 0.6 mM for 12 and 24 h. Furthermore, ADSCs-CM could protect SH-SY5Y cells against hypoxic condition more intensively at all CoCl₂ concentrations and various incubation incubation periods (P<0.01 and P<0.001, respectively). However, there were no significant differences between ADSCs-CM and DPSCs-CM. Also, ADSCs-CM and DPSCs-CM could considerably reduce the LDH release and apoptotic cells when SH-SY5Y cells were exposed to 0.6 mM CoCl₂ for 24 h.
Conclusion: The study indicated that both ADSCs-CM and DPSCs-CM have neuroprotective effects on hypoxic SH-SY5Y cells through reduction of apoptotic cells and release of LDH.

---

     Background and purpose: Tissue engineering and cell therapy, as promising therapies, provide the opportunity to repair bone lesions and defects. Combined scaffolds, synthetic and natural polymers can provide a suitable structure for differentiation of Wharton Jelly mesenchymal stem cells (WJ-MSCs) into bone. In current study, the effect of lyophilized blood growth factors in promoting the process of osteogenesis is important.
Materials and methods: In this study, PCL-Gel nanofiber scaffolds were prepared by electrospinning method. To prepare membrane, blood was collected from volunteers and its growth factors were extracted and loaded on nanofiber layers. They were then placed in a freezer at -20°C for three days and finally in a freezer for 48 hours. They were structurally examined by electron microscopy and sterilized by gamma rays. WJ-MSCs were then cultured on scaffolding. MTT and Real-Time PCR tests were performed to evaluate cell viability and expression of ALP, RUNX2, COLX, and COLI genes in the designed scaffold.
Results: Findings showed that the viability, growth, proliferation, and the expression of WJ-MSCs and osteogenic-specific genes were significantly higher at high concentration of blood derived growth factors/ polycaprolactone /gelatin-scaffold combination (P<0.001).
Conclusion: High concentration of blood derived growth factors in designed scaffolds facilitated the osteogenesis process.

---

Background and purpose: Spermatogenesis is a well-organized process that is influenced by a variety of factors. Alkaline phosphatase, and Gfra1, Lin28, and Sall4 genes are among the key players in this interconnected process. This study aimed to investigate the expression levels of Gfra1, Lin28, and Sall4 genes in embryonic, spermatogonial, and embryonic stem-like (ES-like) cells in mice.
Materials and methods: First, spermatogonial stem cells were isolated, and then mouse embryonic stem cells and ES-like cells were prepared. Alkaline phosphatase expression was determined by alkaline phosphatase staining. After that, a bioinformatics analysis of directly related genes was done. Then the expression levels of the Gfra1, Lin28, and Sall4 genes were measured using Fluidigm PCR, and immunohistochemistry was done for Gfra1 expression.
Results: In pluripotent stem cells and germ cells, the expression levels of Gfra1, Lin28, and Sall4 genes were found to be significantly positive (P<0.05). Alkaline phosphatase was also expressed positively in germ stem cells and pluripotent stem cells, but not in Sertoli cells or fibroblasts, indicating that this enzyme is exclusive to these cell types.
Conclusion: Because of their interaction and expression in pluripotent stem cells and germ cells, alkaline phosphatase, Gfra1, Lin28, and Sall4 genes can be employed as detecting markers for these cell lines.
 

---

Hair loss is a common hair disorder in human population. It affects quality of life and there are ongoing attempts to find permanent treatment for this condition. But, today there is no completely safe and protective treatment for all. Hair follicle stem cells are alive, but quiescence in androgenetic alopecia and are potentially active and can proliferate and differentiate, then regenerate hair follicles. Currently, research is more focussed on mesenchymal stem cells, hair follicle progenitor cells, and dermal papilla cells implantation which act like hair follicle stem cells. Therefore, activating the hair follicle stem cells signaling pathways such as Wnt/β-catenin can stimulate these cells for proliferation and differentiation and lead to hair regeneration. Nowadays, this treatment strategy has shown promising horizon to treat androgenetic alopecia. This paper reviews latest findings in treatment of androgenetic alopecia using re-activation Wnt/β-catenin signaling pathway in hair follicle stem cells.
 

---

 Background and purpose: Human pluripotent stem cells (hPSCs) with the ability to differentiate into adult cells have provided a new perspective for treatment of some diseases. But, the efficiency of differentiation methods to generate hematopoietic progenitor cells (HPCs) is faced with multiple challenges. In the present study, we investigated the formation of hemato-endothelial-like structures and HPCs from hPSCs.
Materials and methods: To generate hemato-endothelial structures and HPCs, in first method, three-dimensional aggregations of hPSCs were co-cultured on OP9 cells. In second and third methods, embryoid bodies (EBs) were differentiated spontaneously or directly in the culture medium containing BMP4, bFGF, SCF, and VEGF. In differentiation process, cell morphology was evaluated by microscopic observation and the expression of CD34 and CD45 markers were analyzed by flow cytometry. In addition, the ability of HPCs to differentiate into various types of blood cells was evaluated using colony formation assay, Wright-Giemsa staining, and CD86 immunofluorescence staining.
Results: Findings showed that all three methods generated cells with endothelial-like morphology and HPCs. These cells were able to proliferate, form cell clusters with different efficiency and differentiate into erythroid cells, macrophages, and dendritic cells.
Conclusion: The use of cultured cells in defined conditions without a feeder layer is preferred in cell therapy. Therefore, despite the ability of all these methods to generate HPCs, direct differentiation of EBs under defined conditions is a better option for generatingHPCs from hPSCs.
 

---

Background and purpose: Dental pulp stem cells (DPSCs) with the ability to differentiate into different types of adult cells have provided a new vision in the treatment of various diseases. However, different challenges remain with differentiation methods to produce insulin-producing cells (IPCs). In the present study, the differentiation of dental pulp stem cells into IPCs using platelet-rich plasma (PRP) has been addressed.
Materials and methods: In this experimental study for generation of IPCs, DPSCs were cultured in the first step, and then to confirm the characteristics of stem cells, the morphology of stem cells was confirmed by microscopic observation, and then the expression of CD90 and CD105 markers assessed by flow cytometry. After confirming the pluripotency of the stem cells, a 18days differentiation protocol into IPCs began. Following that, pancreatic endocrine genes such as insulin, pdx1, glut2 and glucagon were analyzed using Real-Time PCR. Insulin and C-peptide release was assessed using ELISA. To investigate the effect of platelet-rich plasma on cell viability, the MTT test was performed.
Results: Results showed that the use of the above-mentioned differentiation protocol, in the differentiation group containing PRP, the cells resembled Langerhans islet clusters. The expression of pancreatic endocrine genes in the differentiation group containing PRP was significantly higher than the other groups. Additionally, in two differentiation groups with and without PRP, the cells responded differently to glucose concentrations compared to insulin and C-peptide release. Furthermore, the rate of cell proliferation in the differentiation group containing PRP was higher than the other groups.
Conclusion: Based on the availability of dental pulp stem cells and PRP, the absence of invasive methods for obtaining these resources, as well as the low immunogenicity due to the use of the patient's tissues, seems to be appropriate method for producing insulin-secreting cells.
 

---

Background and purpose: The osteogenic differentiation of MSCs plays a key role in bone tissue engineering and regenerative medicine, making it essential to explore natural compounds that may enhance this process. This aim this study was to investigate the osteogenic potential of both aqueous and alcoholic extracts derived from the root of Rubia tinctorum on rat bone marrow-derived mesenchymal stem cells (BM-MSCs).
Materials and methods: Aqueous and alcoholic extracts were prepared from the root of Rubia tinctorum. Rat BM-MSCs were isolated and cultured and their osteogenic differentiation was assessed in the presence of varying concentrations of the extracts using alizarin red staining and alkaline phosphatase (ALP) activity. Cytotoxic effect of root extract of Rubia tinctorum was evaluated using MTT assay.
Results: The MTT assay demonstrated that the root extract of Rubia tinctorum was cytotoxic at 1000 µg/ml. Additionally, the alizarin red assay showed that the most significant color intensity at 1 and 10 µg/ml concentrations on the 14th day. In terms of bone differentiation induction which measured via the alkaline phosphatase test, both the aqueous and alcoholic extracts at 1 and 10 µg/ml concentrations led to substantial increases (14.9- and 14.57-fold for the aqueous extract and approximately 7.37- and 7-fold for the alcoholic extract, respectively) in alkaline phosphatase activity compared to the negative control.
Conclusion: This study sheds light on the osteogenic effects of Rubia tinctorum root extracts, emphasizing their potential as natural agents in promoting bone regeneration and tissue engineering applications. Further investigations into the underlying molecular mechanisms and in vivo studies are warranted to comprehensively evaluate the feasibility of utilizing these extracts for therapeutic bone regeneration purposes
 

---

 Background and purpose: Endothelial progenitor cells (EPCs) and mesenchymal stem cells (MSCs) have useful effects in treatment of sepsis, but the effects of co-injection of EPC and MSC for the treatment of sepsis have not yet been investigated. This study investigated the therapeutic effect of MSC+EPC in reducing the systemic inflammatory response in lipopolysaccharide (LPS) induced sepsis in mice.
Materials and methods: In this experimental study, mouse bone marrow MSCs and EPCs were isolated and their surface markers were investigated using flow cytometry. Four groups of mice (n= 12 per group) were included in the study. The control group had no treatment, group II received LPS intraperitoneally, group III received PBS buffer after LPS inoculation, and group IV received EPCs+MSCs after LPS inoculation. The serum and tissue levels of IL-1β, TNF-α, IL-6, and IL-10, and the serum levels of CRP and hepatic enzymes were determined by commercial kits. One-way ANOVA and post hoc test were used for comparison between groups.
Results: The surface markers of MSCs and EPCs were confirmed by flow cytometry. The co-injection of MSC+EPC significantly decreased the levels of pro-inflammatory cytokines (P<0.001), increased the concentration of anti-inflammatory cytokines (P<0.001), and increased the survival rate (P<0.01). Mice treated with MSC+EPC represented a significant decrease in liver enzymes (P<0.05), pulmonary edema (P<0.01), and CRP level (P<0.05) compared to the mice with LPS-induced sepsis.
Conclusion: Co-injection of MSC and EPC leads to a reduction of the inflammatory response. This study provides promising results for the treatment of sepsis.
 

---

Background and purpose: Degenerative joint disease, especially osteoarthritis (OA), is a global disease characterized by the destruction of articular cartilage, and subchondral bone. It is estimated that about 250 million people currently suffer from cartilage defects. So far, no definite and standard treatment method for OA has been reported. Recently, cell-based therapeutic techniques have been considered one of the best therapeutic strategies for the long-term treatment of articular cartilage diseases. However, many challenges include the large scale of cells required, thus the cell-free approaches are novel tools for cartilage defect treatment. For instance, extracellular vesicles (EVs) secreted by various cells such as MSCs are in charge of the therapeutic effects of stem cells. Therefore, recently EVs have advanced as powerful cell-free transfer tools, due to their high physicochemical strength and biocompatibility.
Materials and methods: This study is a review study that summarizes the preclinical and clinical studies that used EV-derived from different cell sources and investigates their effectiveness in treating cartilaginous tissue lesions. Current studies used small or large animal models with experimental critical size defects in knee articular cartilage to examine the effectiveness of EVs derived from MSCs. The EVs were isolated from cell sources such as adipose-derived MSCs, Bone marrow-derived MSCs, or transgenic cells. In addition, EV isolation techniques as a main challenge in studies using EVs to treat OA, specifically described in the current study. We also showed EVs isolated from each cell have unique features such as anti-inflammatory, differentiation, and therapeutic properties. We explain recent studies that use EVs as a drug carrier such as small molecules, and microRNA bioactive factors. In addition, the isolation techniques of EVs and their characterization are other challenges that we explain.
Results: Recent studies have shown that EVs isolated from different sources inhibit the progression of OA. Also, the results of some studies indicate the ability of EVs to repair injured cartilage. Many studies showed that in critical size defects of cartilage, the use of EVs needs scaffolds. Several studies have investigated the challenges of EV release and the required EV dose based on the size of the lesion. EVs are rapidly emerging as novel therapeutic approaches for treating cartilage lesions and OA. Despite many advances in cell therapy and promising results reported in numerous disease models, the use of cells especially genetically modified cells has limitations in regenerative medicine. It is worth noting that the use of EVs derived from stem cells or transgenic cells has no harm to the human body. As a result, therapeutic EVs have been introduced as a new therapeutic approach that does not have the same potential risks as cells.
Conclusion: Despite the positive results of EV in the treatment of cartilaginous lesions, it appears that the EV therapeutic barrier requires further testing in larger animal models before clinical trials. For instance, the regeneration of critical-size lesions requires the use of EVs incorporated by suitable scaffolds under dynamic conditions. Therefore, the fundamental questions to be considered are: How to use EVs as a nanoparticle instead of stem cells in combination with tissue engineering methods? What are the biological properties of EVs? What doses of EVs have the mechanistic potential for the treatment of different sizes of cartilage lesions and how EVs are stable in lesions? What is the role of EVs in the homeostasis and pathogenesis of junctions?
 

---

Background and purpose: Menstrual blood-derived mesenchymal stem cells (MB-MSCs) expressing CD44, CD90, and CD105 markers were recently introduced. These cells are a good source of stem cells for research and use in regenerative medicine due to uncomplicated collection without invasive surgical intervention or ethical issues. Cell culture is one of the most essential techniques in molecular cell biology, and the culture medium is the most critical component. Serum is one of the crucial components of the culture medium and a protective solution. One of the most common serums used in cell culture is fetal bovine serum (FBS). This serum is an unknown mixture and can contain unfavorable factors such as endotoxin, mycoplasma, viral contaminants, or prion proteins. Therefore, there is a need for a human substitute for FBS that can be utilized for clinical applications. In this study, a humanized protocol utilizing autologous serum (AS) instead of FBS is tested to investigate the expansion of MB-MSCs and exosomes released from these cells.
Materials and methods: A menstrual blood sample was collected from the donor on the second day of menstruation. Autologous serum samples were also collected to prepare the culture medium. First, mesenchymal stem cells were extracted from menstrual blood and then cultured in an environment enriched with autologous serum at different concentrations of 10%, 15%, and 20%. The investigation was done on the cells obtained in the third passage. Cultured cells in autologous serum were analyzed regarding expansion and expression of mesenchymal surface markers (CD73 and CD105). An inverted microscope was used to study the expansion, and flow cytometry was used to investigate the expression of mesenchymal markers. Also, in the next step, the culture medium of cultured cells in autologous serum was collected for exosome isolation. Exosome isolation was done by a three-step combination method of sedimentation, size exclusion chromatography with CL-2B sepharose resin, and making it concentrated. Finally, the purified exosomes were analyzed regarding morphology, size, and expression of CD9, CD63, and CD81 markers. Transmission electron microscope (TEM), dynamic light scattering (DLS), and flow cytometry analysis were operated, respectively.
Results: According to the observations, cell expansion was observed in all three concentrations of autologous serum, and the most favorable results were marked in the concentration of 15%. In addition, flow cytometry results indicated the expression of mesenchymal markers CD73 and CD105 in cells cultured with 15% autologous serum. Exosomes were isolated from the culture medium of mesenchymal stem cells cultured with autologous serum, and their characteristics were investigated using dynamic light scattering, transmission electron microscope, and flow cytometry techniques. The size of the exosomes ranged from 30-150 nm, and their morphology was cup-shaped. The expression of exosome markers such as CD9, CD63, and CD81 was also confirmed.
Conclusion: As a result, with the increase of using mesenchymal stem cells in regenerative medicine, it is imperative to ensure the safety of the process and materials used in this field. Therefore, autologous serum can be a suitable option for the culture of mesenchymal stem cells derived from menstrual blood.