The effects of intraovarian injection of autologous menstrual blood-derived mesenchymal stromal cells on pregnancy outcomes in women with poor ovarian response.

BACKGROUND: Assisted reproduction faces a significant obstacle in the form of poor ovarian response (POR) to controlled ovarian stimulation. To address this challenge, mesenchymal stem cell therapy has been proposed as a potential treatment for female infertility and/or restoration of ovarian function in POR women. Our previous research has demonstrated that menstrual blood-derived-mesenchymal stromal cells (MenSCs) injected into the ovaries of women with POR can increase pregnancy rates. The objective of this study was to examine whether MenSC therapy could enhance ovarian reserve parameters and pregnancy outcomes in a larger population of individuals with POR. METHOD: This study consisted of 180 infertile individuals with POR who declined oocyte donation. Participants were divided into two groups: those who received bilateral MenSCs intraovarian injection and those who received no intervention. Our primary aim was to compare the rates of spontaneous pregnancy between the two groups, followed by an investigation of any alterations in the ovarian reserve parameters, such as serum FSH, AMH, and AFC levels, as well as the ICSI/IVF outcomes, in both groups of participants. RESULTS: The MenSC therapy exhibited a favourable tolerability profile and did not raise any safety concerns. Following the 2-month follow-up period, women who received MenSC treatment demonstrated a significantly higher rate of spontaneous pregnancy (P < 0.005) and an improvement in anti-Müllerian hormone (AMH) levels (P = 0.0007) and antral follicle count (AFC) (P < 0.001), whereas the control group demonstrated a considerable decline in these parameters (Both P < 0.001). The MenSC therapy led to a greater number of mature oocytes and embryos among women who underwent ICSI/IVF. Our age subgroup analysis demonstrated a significant difference in the number of spontaneous pregnancies and ICSI/IVF outcomes between the treatment and control groups only among individuals below 40 years of age. CONCLUSION: The results of our study indicate that MenSCs treatment may be a viable option for treating women experiencing POR. However, in order to be widely implemented in clinical practice, the clinical effectiveness of MenSCs therapy will need to be established through rigorous prospective randomized clinical trials. TRIAL REGISTRATION: ClinicalTrials.gov Identifier: NCT05703308. Registered 01/26/2023, retrospectively registered, https://clinicaltrials.gov/ct2/show/NCT05703308 . IRCT, IRCT20180619040147N4. Registered 08/01/2020.

Application of Menstrual Blood Derived Stromal (stem) Cells Exert Greater Regenerative Potency Than Fibroblasts/Keratinocytes in Chronic Wounds of Diabetic Mice.

Background: In this study we differentially showed the effects of cell-seeded bilayer scaffold wound dressing in accelerating healing process in diabetic ulcers that still remains as a major clinical challenge. The aim of the study was to compare immunomodulatory and angiogenic activity, and regenerative effect differences between Menstrual blood-derived Stem Cells (MenSCs) and foreskin-derived keratinocytes/fibroblasts. Methods: The streptozotocin-induced diabetic mice model was developed in male C57/BL6 mice. A bilayer scaffold was fabricated by electrospining silk fibroin nano-fibers on human Amniotic Membrane (AM). Dermal fibroblasts and keratinocyte isolated from neonatal foreskin and MenSCs were isolated from the menstrual blood of healthy women. The diabetic mice were randomly divided into three groups including no treatment group, fibroblast/keratinocyte-seeded bilayer scaffold group (bSC+FK), and MenSCs-seeded bilayer scaffold group. The healing of full-thickness excisional wounds evaluations in the diabetic mice model in each group were evaluated at 3, 7, and 14 days after treatment. Results: The gross and histological data sets significantly showed wound healing promotion via re-epithelialization and wound contraction along with enhanced regeneration in MenSCs-seeded bilayer scaffold group with the most similarity to adjacent intact tissue. Immunofluorescence staining of mouse skin depicted a descending trend of type III collagen along with the higher expression of involucrin as keratinocyte marker in the MenSCs-seeded bilayer nanofibrous scaffold group in comparison with other treatment groups from day 7 to day 14. Moreover, higher levels of CD31 and von Willebrand factor (VWF), and also a higher ratio of M2/M1 macrophages in association with higher levels of the neural marker were observed in the bSC+MenSCs group in comparison with bSC+FK and no treatment groups. Conclusion: Healing symptoms in wounds dressed with keratinocyte/fibroblast-seeded bilayer scaffold was significantly lower than MenSCs-seeded bilayer scaffold done on impaired diabetic wound chronicity.

Higher Improvement of Cardiac Function Following Myocardial Infarction using Menstrual Blood Stromal/Stem Cells (MenSCs) Suspended in Conditioned Medium versus Conditioned Medium Alone in Rat Model.

Background: To evaluate the efficiency of Menstrual blood Stromal/Stem Cells (MenSCs) administration in Myocardial Infarction (MI), the effects of MenSCs and their derived conditioned Medium (CM) on cardiac function in MI rat model was assessed. Methods: Animals were divided into four groups including sham group, MI group, MenSCs derived CM group (CM group), and MenSCs suspended in CM (MenSCs+CM) group. The injection of different groups was carried out 30 min after ligation of left anterior descending coronary artery into the infarct border zone. Results: The results showed a significant reduction in scar size after injection of MenSCs+CM compared to MI group. Ejection fraction and fractional shortening of MenSCs+CM group were higher than CM and MI group at day 28. Administration of MenSCs+CM led to much more survival of cardiomyocytes, and prevention of meta-plastic development. Moreover, human mitochondrial transfer from MenSCs to cardiomyocytes was seen in group treated by MenSCs+CM. Indeed, MenSCs+CM treatment evoked nuclear factor-κB (NF-κB) down-regulation more than other treatments. Conclusion: MenSCs+CM treatment could significantly ameliorate cardiac function by different mechanisms including inhibition of cartilaginous metaplasia, inhibition of NF-κB and mitochondrial transfer.

Systemic delivery of menstrual blood stem cells is more effective in preventing remote organ injuries following myocardial infarction in comparison with bone marrow stem cells in rat.

Objectives: Remote organ injury is a phenomenon that could happen following myocardial infarction (MI). We evaluated the potency of menstrual blood stromal (stem) cells (MenSCs) and bone marrow stem cells (BMSCs) to alleviate remote organ injuries following MI in rats. Materials and Methods: 2 × 106 MenSCs or BMSCs were administrated seven days after MI induction via the tail vein. Four weeks after cell therapy, activities of aspartate aminotransferase (AST), urea, creatinine, and Blood Urea Nitrogen (BUN) were evaluated. The level of tumor necrosis factor-α (TNF-α), interleukin (IL)-1ß, and IL-6 were determined by ELISA assay. The expression of Nuclear Factor-κB (NF-κB) was evaluated by immunohistochemical staining. Apoptosis activity and tissue damage were also determined by TUNEL and H&E staining, respectively. Results: MenSCs and BMSCs administration caused a significant reduction in AST, urea, and BUN levels compared with the MI group. In addition, systemic injection of MenSCs significantly decreased the IL-1ß level compared with BMSCs and MI groups (P<0.05 and P<0.01 respectively). Apoptosis in injured kidneys was noticeably diminished in MenSCs-treated rats compared with BMSCs administrated and MI groups (P<0.05 and P<0.05, respectively). In hepatic tissue, limited numbers of TUNEL-positive cells were detected in all groups. Interestingly, MenSCs therapy evoked inhibition of NF-κB in the kidney strikingly. Although, no significant NF-κB expression was observed in hepatic tissue in any group (P>0.05). Conclusion: MenSCs are probably more protective than BMSCs on remote organ injuries following MI via decreasing cell death and immunoregulatory properties.

Intraovarian Administration of Autologous Menstrual Blood Derived-Mesenchymal Stromal Cells in Women with Premature Ovarian Failure.

BACKGROUND: Premature ovarian failure (POF) is a well-known cause of infertility, particularly in women under the age of 40. POF is also associated with elevated gonadotropin levels, amenorrhea and sex-hormone deficiency. AIM OF THE STUDY: In this study, the therapeutic potential of autologous mesenchymal stromal cells obtained from menstrual blood (Men-MSCs) for patients with POF was evaluated. METHODS: 15 POF patients were included in the study. The cultured Men-MSCs were confirmed by flow cytometry, karyotype, endotoxin and mycoplasma and were then injected into the patients' right ovary by vaginal ultrasound guidance and under general anesthesia and aseptic conditions. Changes in patients' anti-Müllerian hormone (AMH), antral follicle count (AFC), follicle-stimulating hormone (FSH), luteal hormone (LH), and estradiol (E2) levels, as well as general flushing and vaginal dryness were followed up to one year after treatment. RESULTS: All patients were satisfied with a decrease in general flushing and vaginal dryness. 4 patients (2.9%) showed a spontaneous return of menstruation without additional pharmacological treatment. There was a significant difference in AFC (0 vs. 1 ± 0.92 count, p value ≤0.001%), FSH (74 ± 22.9 vs. 54.8 ± 17.5 mIU/mL, p-value ≤0.05%), E2 (10.2 ± 6 vs. 21.8 ± 11.5 pg/mL p-value ≤0.01%), LH (74 ± 22.9 vs. 54.8 ± 17.5 IU/L,p-value ≤0.01%) during 3 months post-injection. However, there were no significant changes in AMH (p-value ≥0.05%). There were also no significant differences in assessed parameters between 3 and 6 months after cell injection. CONCLUSION: According to the findings of this study, administration of Men-MSCs improved ovarian function and menstrual restoration in some POF patients.

Greater angiogenic and immunoregulatory potency of bFGF and 5-aza-2'-deoxycytidine pre-treated menstrual blood stem cells in compare to bone marrow stem cells in rat model of myocardial infarction.

BACKGROUND: This study is designed to compare the menstrual blood stem cells (MenSCs) and bone marrow stem cells (BMSCs)-secreted factors with or without pre-treatment regimen using basic fibroblast growth factor (bFGF) and 5-aza-2'-deoxycytidine (5-aza) and also regenerative capacity of pre-treated MenSCs and/or BMSCs in a rat model of myocardial infarction (MI). METHODS: BMSCs and MenSCs were pre-treated with bFGF and 5-aza for 48 h and we compared the paracrine activity by western blotting. Furthermore, MI model was created and the animals were divided into sham, MI, pre-treated BMSCs, and pre-treated MenSCs groups. The stem cells were administrated via tail vain. 35 days post-MI, serum and tissue were harvested for further investigations. RESULTS: Following pre-treatment, vascular endothelium growth factor, hypoxia-inducible factor-1, stromal cell-derived factor-1, and hepatocyte growth factor were significantly increased in secretome of MenSCs in compared to BMSCs. Moreover, systemic administration of pre-treated MenSCs, leaded to improvement of cardiac function, preservation of myocardium from further subsequent injuries, promotion the angiogenesis, and reduction the level of NF-κB expression in compared to the pre-treated BMSCs. Also, pre-treated MenSCs administration significantly decreased the serum level of Interleukin 1 beta (IL-1ß) in compared to the pre-treated BMSCs and MI groups. CONCLUSIONS: bFGF and 5-aza pre-treated MenSCs offer superior cardioprotection compare to bFGF and 5-aza pre-treated BMSCs following MI.

Safety and efficacy study of allogeneic human menstrual blood stromal cells secretome to treat severe COVID-19 patients: clinical trial phase I & II.

BACKGROUND: Cell-free Mesenchymal stromal cells (MSCs) have been considered due to their capacity to modulate the immune system and suppress cytokine storms caused by SARS-CoV-2. This prospective randomized double-blind placebo-controlled clinical trial aimed to assess the safety and efficacy of secretome derived from allogeneic menstrual blood stromal cells (MenSCs) as a treatment in patients with severe COVID-19. METHODS: Patients with severe COVID-19 were randomized (1:1) to either MenSC-derived secretome treatment or the control group. Subjects received five intravenous infusions of 5 mL secretome or the same volume of placebo for five days and were monitored for safety and efficacy for 28 days after treatment. Adverse events, laboratory parameters, duration of hospitalization, clinical symptom improvement, dynamic of O2 saturation, lymphocyte number, and serial chest imaging were analyzed. RESULTS: All safety endpoints were observed without adverse events after 72 h of secretome injection. Within 28 days after enrollment, 7 patients (50%) were intubated in the treated group versus 12 patients (80%) in the control group. Overall, 64% of patients had improved oxygen levels within 5 days of starting treatment (P < 0.0001) and there was a survival rate of 57% in the treatment group compared to 28% in the control group was (P < 0.0001). Laboratory values revealed that significant acute phase reactants declined, with mean C-reactive protein, ferritin, and D-dimer reduction of 77% (P < 0.001), 43% (P < 0.001), and 42% (P < 0.05), respectively. Significant improvement in lymphopenia was associated with an increase in mean CD4+ and CD8+ lymphocyte counts of 20% (P = 0.06) and 15% (P < 0.05), respectively. Following treatment, percentage of pulmonary involvement showed a significant improvement in the secretome group (P < 0.0001). This improvement differed significantly between survivors and those who were dying (P < 0.005). CONCLUSIONS: For the first time, this study demonstrated that in hospitalized patients with severe COVID-19, therapy with MenSCs-derived secretome leads to reversal of hypoxia, immune reconstitution, and downregulation of cytokine storm, with no adverse effects attributable to the treatment. Given these outcomes, it may be possible to use this type of treatment for serious inflammatory lung disease with a mechanism similar to COVID-19 in the future. However, it is necessary to evaluate the safety and efficacy of MenSCs-derived secretome therapy in clinical trials on a larger population of patients. TRIAL REGISTRATION: ClinicalTrials.gov Identifier: NCT05019287. Registered 24AGUEST 2021, retrospectively registered, https://clinicaltrials.gov/ct2/show/record/NCT05019287 . IRCT, IRCT20180619040147N6. Registered 04/01/2021.

The remarkable effect of menstrual blood stem cells seeded on bilayer scaffold composed of amniotic membrane and silk fibroin aiming to promote wound healing in diabetic mice.

INTRODUCTION: Impaired chronic wound healing frequently occurs in diabetic patients. We hypothesized that menstrual blood-derived mesenchymal stem cells (MenSCs) in combination with bilayer scaffold consisted of human amniotic membrane (AM) and electrospun silk fibroin nanofibers could potentially promote wound healing in diabetic mice. METHODS & METHODS: Two bilateral full-thickness wounds were created on dorsal skin of type-1 diabetic mice model and animals were equally divided in four groups including: no-treatment group (NT), amniotic membrane treated group (AM), bilayer scaffold treated group (bSC), and MenSCs-seeded bilayer scaffold treated group (bSC + MenSCs). Wound healing evaluations were performed at 3, 7, and 14 days after their treatment. The wound healing was analyzed by macroscopic and microscopic evaluations, and immunofluorescence staining of involucrin (IVL), type III collagen, CD31/ von Willebrand factor (vWF), and PGP9.5 were performed. Furthermore, number of neutrophils and macrophages and subpopulation of macrophages were assessed. In addition, the expression of Egr2, Mmp9, CXCL12, IDO1, Ptgs2 and VEGFA transcripts involved in wound repair were also analyzed. RESULTS: After 14 days, the best epidermal and dermal regeneration belonged to the cases received bSC + MenSCs as wound dressing. Moreover, the wound healing was typically faster in this group compared to other groups. Immunofluorescence evaluation represented higher levels of CD31 and VWF, higher ratio of M2/M1 macrophages, greater expression of IVL, and higher levels of the PGP9.5 in the bSC + MenSCs group in comparison with other groups. Expression analysis of assessed genes also supported assumption of more regeneration and healing in the bSC + MenSCs group versus other groups. CONCLUSION: These results indicate that enhanced immunomodulatory and reparative properties of MenSCs in conjunction with bilayer scaffold specified this cellular skin substitute for modulating wound chronicity and contribution to resolution of wound healing process in diabetic ulcer.

Safety and efficacy of engineered tissue composed of silk fibroin/collagen and autologous chondrocytes in two patients with cartilage defects: A pilot clinical trial study.

BACKGROUND: The objective of this pilot clinical trial study was to evaluate safety and effectiveness of the newly engineered tissue composed of autologous chondrocytes and collagen/fibroin scaffold in repair of osteochondral defects. METHODS: We implemented a pilot clinical study in two patients with knee osteochondral lesions using engineered tissue composed of scaffold and autologous chondrocytes. Patients were clinically evaluated using the International Repair Cartilage Society score and magnetic resonance imaging (MRI) for one year. RESULTS: Improved clinical outcomes and objective scores indicated a normal or nearly normal knee in both patients. International Knee Documentation Committee score was upgraded from 34.5 at baseline to 72.4 in the first patient, and 28.7 to 81.6 in the second patient. Visual analogue scale, showing the suffering pain score, was lowered from 8 to 0 in both patients, Western Ontario and McMaster Universities Osteoarthritis Index score representing the physical ability of the patients was changed from 68.1 to 87.1 in Patient 1 and 58.3 to 87.1 in Patient 2, the knee function score, related to the functional ability of the knee, was improved from 70 to 100 in the first patient and from 45 to 91 in the second patient. MRI showed great coverage and integration of the graft in patients, with no effusion, decreased edema and cartilage formation signals. CONCLUSIONS: The functional and clinical outcomes alongside MRI data showed promising results for regenerating osteochondral defects. A randomized clinical trial study is required to confirm feasibility of this novel engineered tissue in repair of osteochondral defects.

Improvement of Pregnancy Rate and Live Birth Rate in Poor Ovarian Responders by Intraovarian Administration of Autologous Menstrual Blood Derived- Mesenchymal Stromal Cells: Phase I/II Clinical Trial.

The study was aimed to evaluate the safety and efficacy of cell therapy using autologous menstrual blood derived- mesenchymal stromal cells (Men-MSCs) in fertility potential of poor ovarian responders (PORs). POR women were divided into mesenchymal stroma cell (MSC) therapy (n = 15) and routine ICSI (n = 16) groups. The cultured Men-MSCs were autologously injected into left ovary of MSC group after approval by flow cytometry, karyotyping, endotoxin, sterility and mycoplasma tests. Changes in anti-Mullerian hormone (AMH), antral follicles count (AFC), oocytes and embryos number, clinical pregnancy rate and live birth rate were followed in both groups up to one year after treatment. 4 of 15 participants in MSC group got naturally pregnant during 3 months after cell administration, in contrast to no natural conception in control group (P = 0.04). The mean AMH level did not significantly differ with that of previous cycle or control group. Although mean AFC and oocytes number in MSC group did not indicate considerable difference with those of control group, raise of these parameters in comparison with previous cycle was significant (both P = 0.01). Nonetheless, oocyte fertilization rate and embryo number in MSC group were higher than control group (P = 0.04 and P = 0.008, respectively). Altogether, 7 of 15 women in MSC group and 2 of 16 women in routine ICSI group had clinical pregnancy that resulted in 5 live births in main group and one birth in control group. In conclusion, cell therapy using Men-MSCs could be considered as a potential treatment to restore fertility capability of POR women.The trial registration number (TRN): IRCT20180619040147N2.Date of registration: 2018-08-21.

Comparative effectiveness of three-dimensional scaffold, differentiation media and co-culture with native cardiomyocytes to trigger in vitro cardiogenic differentiation of menstrual blood and bone marrow stem cells.

The main purpose of this study was to find effectiveness of 3D silk fibroin scaffold in comparison with co-culturing in presence of native cardiomyocytes on cardiac differentiation propensity of menstural blood(MenSCs)-versus bone marrow-derived stem-cells (BMSCs). We showed that both 3D fibroin scaffold and co-culture system supported efficient cardiomyogenic differentiation of MenSCs and BMSCs, as judged by the expression of cardiac-specific genes and proteins, Connexin-43, Connexin-40, alpha Actinin (ACTN-2), Tropomyosin1 (TPM1) and Cardiac Troponin T (TNNT2). No significant difference (except for higher expression of ACTN-2 in co-cultured MenSCs) was found between differentiation potential of the cells cultured in 3D fibroin scaffold and co-culture system. Collectively, our results imply that inductive signals served by biological factors of native cardiomyocytes to trigger cardiogenic differentiation of stem-cells may be efficiently provided by natural and biocompatible 3D fibroin scaffold suggesting the usefulness of this construct for cardiac tissue engineering.

A Novel Monoclonal Antibody Against a Synthetic Peptide from ß-Actin can React with its Corresponding Protein.

Actin is one of the most widely studied structural and multifunctional housekeeping proteins in eukaryotic cells with important roles in many cell functions. Antibodies against ß-actin and other housekeeping gene-encoded proteins are used as internal loading controls in Western blot analyses. The aim of this study was to produce a monoclonal antibody (mAb) against a synthetic peptide derived from N-terminal region of ß-actin and to study its reactivity with different organisms. A synthetic peptide, derived from ß-actin, was designed and used to produce a mAb by hybridoma technology. The produced antibody (clone 4E5- A10) was purified by an affinity chromatography column followed by characterization of purified mAb using SDS-PAGE, ELISA and Western blot. Our results showed that 4E5-A10 was an IgM and had desired purity and excellent reactivity with the immunizing peptide with an affinity constant of 2.7x10(8) M(-1)>. It could detect a band of about 45 kDa, corresponding to ß-actin, in Western blot. Furthermore, it could react in a more sensitive manner and with a wider range of organisms than a known commercial anti ß-actin antibody. Our data suggest that 4E5-A10 can act as a sensitive probe for detection of ß-actin as an internal loading control, for a wide range of organisms, in Western blot analyses.

High placenta-specific 1/low prostate-specific antigen expression pattern in high-grade prostate adenocarcinoma.

BACKGROUND: The scarcity of effective therapeutic approaches for prostate cancer (PCa) has encouraged steadily growing interest for the identification of novel antigenic targets. Placenta-specific 1 (PLAC1) is a novel cancer-testis antigen with reported ectopic expression in a variety of tumors and cancer cell lines. The purpose of the present study was to investigate for the first time the differential expression of PLAC1 in PCa tissues. METHODS: We investigated the differential expression of PLAC1 in PCa, high-grade prostatic intraepithelial neoplasia (HPIN), benign prostatic hyperplasia (BPH), and nonneoplastic/nonhyperplastic prostate tissues using microarray-based immunohistochemistry (n = 227). The correlation of PLAC1 expression with certain clinicopathological parameters and expression of prostate-specific antigen (PSA), as a prostate epithelial cell differentiation marker, were investigated. RESULTS: Placenta-specific 1 (PLAC1) expression was increased in a stepwise manner from BPH to PCa, which expressed highest levels of this molecule, while in a majority of normal tissues, PLAC1 expression was not detected. Moreover, PLAC1 expression was positively associated with Gleason score (p ≤ 0.001). Interestingly, there was a negative correlation between PLAC1 and PSA expression in patients with PCa and HPIN (p ≤ 0.01). Increment of PLAC1 expression increased the odds of PCa and HPIN diagnosis (OR 49.45, 95 % CI for OR 16.17-151.25). CONCLUSION: Our findings on differential expression of PLAC1 in PCa plus its positive association with Gleason score and negative correlation with PSA expression highlight the potential usefulness of PLAC1 for targeted PC therapy especially for patients with advanced disease.

Immunohistochemical characterization of novel murine monoclonal antibodies against human placenta-specific 1.

Human PLAC1 (placenta-specific 1) is a new member of cancer-testis antigens with 212 amino acids, and its expression is restricted to placenta and at much lower levels to testis. Recently, ectopic expression of the PLAC1 transcript has been demonstrated in a wide range of human tumors and cancer cell lines with a proposed function in tumor cell growth. No monoclonal anti-PLAC1 antibody applicable to immunohis-tochemical staining is available so far. To better understand the PLAC1 expression and localization, we aimed to produce monoclonal antibodies (mAbs) against the extracellular region of PLAC1. Mice were immunized with a synthetic peptide corresponding to the C-terminal 11 amino acids of PLAC1 conjugated with a carrier protein. Hybridomas were produced by standard protocol and screened for positive reactivity by enzyme-linked immunosorbent assay. Reactivity of final two clones was then assessed by Western blotting (WB), immunohistochemistry (IHC), and immunocytochemistry (ICC). Both clones showed a specific immunostaining pattern in human term placenta as the positive control. Reactivity was mostly localized to the cytoplasm of syncytiotrophoblasts. One of the clones showed an excellent staining signal in breast, ovary, and prostate cancer cell lines. Importantly, no reactivity was observed with human lymph node cells or prostate. None of the mAbs were able to detect PLAC1 in Western blot. Based on the present results, these mAbs can be used for detection of PLAC1 in IHC and ICC techniques.

Comparison of the Photobleaching and Photostability Traits of Alexa Fluor 568- and Fluorescein Isothiocyanate- conjugated Antibody.

OBJECTIVE: Synthetic fluorescent dyes that are conjugated to antibodies are useful tools to probe molecules. Based on dye chemical structures, their photobleaching and photostability indices are quite diverse. It is generally believed that among different fluorescent dyes, Alexa Fluor family has greater photostability than traditional dyes like fluorescein isothiocyanate (FITC) and Cy5. Alexa Fluor 568 is a member of Alexa Fluor family presumed to have superior photostability and photobleahing profiles than FITC. MATERIALS AND METHODS: In this experimental study, we conjugated Alexa Fluor 568 and FITC dyes to a mouse anti-human nestin monoclonal antibody (ANM) to acquire their photobleaching profiles and photostability indices. Then, the fluorophore/antibody ratios were calculated using a spectrophotometer. The photobleaching profiles and photostability indices of conjugated antibodies were subsequently studied by immunocytochemistry (ICC). Samples were continuously illuminated and digital images acquired under a fluorescent microscope. Data were processed by ImageJ software. RESULTS: Alexa Fluor 568 has a brighter fluorescence and higher photostability than FITC. CONCLUSION: Alexa Fluor 568 is a capable dye to use in photostaining techniques and it has a longer photostability when compared to FITC.