Characteristics of the human endometrial regeneration cells as a potential source for future stem cell-based therapies: A lab resources study

Background: Human endometrium with consecutive regeneration capability undergoes monthly hormonal changes for probable implantation, which confirms the presence of the cells in the basalis layer known as stem cell. Objective: Previously, we reported the isolation and culture of the mesenchymal-like cells from human endometrium. In this study, we evaluated the biological and stemness characteristics of these cells. Materials and Methods: The characterization of Yazd human endometrialderived mesenchymal stem/stromal cells (YhEnMSCs) was assessed using immunofluorescence (IF) staining for CD105, VIMENTIN, and FIBRONECTIN as markers and RT-PCR for CD166, CD10, CD105, VIMENTIN, FIBRONECTIN, MHCI, CD14, and MHCII genes. Flow cytometry (FACS) was performed for CD44, CD73, CD90, and CD105 markers. Moreover, the differentiation capacity of the YhEnMSCs to the osteoblast and adipocytes was confirmed by Alizarin Red and Oil Red staining. Results: YhEnMSCs expressed CD105, VIMENTIN, FIBRONECTIN, CD44, CD73, and CD90 markers and CD166, CD10, CD105, VIMENTIN, FIBRONECTIN, and MHCI, but, did not express CD14, MHCII. Conclusion: Our data confirm previous reports by other groups indicating the application of endometrial cells as an available source of MSCs with self-renewal and differentiation capacity. Accordingly, YhEnMSCs can be used as a suitable source for cell-based therapies. Key words: Cell-based therapy, Endometrium, Mesenchymal stem/stromal cells, Regenerative medicine, Stem cells, Uterus

Increased expression of stemness genes Rex-1, Oct-4, Nanog, and Sox-2 in women with ovarian endometriosis versus normal endometrium: A case-control study

Background: Endometriosis is a common, chronic inflammatory disease which is defined as an overgrowth of endometrial tissue outside the uterine cavity. The etiology of this disease is complex and multifactorial but there is a strong evidence that supports the presence of endometrial stem cells and their possible involvement in endometriosis. Objective: In this study, we analyzed the mRNA expression of REX-1 stemness gene and reconsidered three other stemness genes SOX-2, NANOG, OCT-4 in women with endometriosis compared to normal endometrium. Materials and Methods: Ten ectopic and ten eutopic tissue samples along with 23 normal endometrium specimens were recruited in this study. The expression levels of OCT-4, NANOG, SOX-2, and REX-1 genes were evaluated by the quantitative real-time polymerase chain reaction. Results: The transcription levels of OCT-4, NANOG, and SOX-2 mRNA were significantly increased in ectopic lesions compared with eutopic and control group (p = 0.041, p = 0.035, p = 0.048), although the REX-1 mRNA increase was not significant between endometriosis and control groups. Also, there were differences in the expression level of these genes in normal endometrium during the menstrual cycles (p = 0.031, p = 0.047, p = 0.031). Conclusion: Based on our data, we confirm the dynamic role of stemness genes in proliferation and growth of normal endometrium during the menstrual cycle and conclude that differential expression n levels of these genes may contribute to the pathophysiology of endometriosis. Key words: Endometriosis, Stemness genes

Biological and physiological characteristics of human cumulus cell in adherent culture condition

Background: Cumulus cells, as oocyte nurse cells, provide a suitable microenvironment with growth factors and cellular interactions required for oocyte maturation. Thus, these cells may serve as a natural niche for in vitro studies of female germ cell development. Cumulus cells may help attain a better understanding of the causes of infertility in women and eventually improve the outcomes of cases that respond poorly to standard infertility treatment. Objective: The aim of this study was to isolate, culture, and investigate the biological characteristics of human cumulus cells. Materials and Methods: In this experimental study, cumulus cells were isolated, cultured, and characterized using reverse transcription-polymerase chain reaction analyses of specific genes including FOXL2, CYP19A1, FSHR, AMHR, and LHR. The presence of vimentin, a structural protein, was examined via immunofluorescent staining. Moreover, levels of anti-mullerian hormone (AMH) and progesterone secretion by cumulus cells were measured with ELISA after 2, 4, 12, 24, and 48 hr of culture. Results: In adherent culture, human cumulus cells expressed specific genes and markers as well as secreted AMH and progesterone into the medium. Conclusion: Cumulus cells secrete AMH and progesterone in an adherent culture and might be applicable for in vitro maturation (IVM) and in vitro gametogenesis (IVG) studies. Key words: Cumulus cells, Conditioned medium, In vitro maturation, In vitro gametogenesis, Niche

Established Yazd human foreskin fibroblast lines (#8, #17, and #18) displaying similar characteristics to mesenchymal stromal cells: A lab resources report

Background: Fibroblasts from different parts of the human body have been used in cell biology, drug discovery and cell therapy studies. One of the most available sources of human fibroblasts is neonatal foreskin. Not only do these cells have woundhealing applications, but they are also the most popular source for pluripotent stem cell biotechnology. Moreover, several studies have indicated that different sources of fibroblasts display similar features to mesenchymal stem cells. Objective: Generation and establishment of new human foreskin fibroblast cell lines called Yazd human foreskin fibroblasts (YhFFs). Materials and Methods: In this lab resources study, the production of 3 YhFF cell lines (YhFF#8, YhFF#17, and YhFF#18) is reported. Their biological features were characterized using immunofluorescence, polymerase chain reaction, and flowcytometry for mesenchymal markers such as fibronectin, vimentin, CD44, CD73, CD90, CD105, and hematopoietic markers CD34 and CD45. Results: The YhFF cell lines were passaged more than 40 times and their normal karyotype was checked using G-binding. Similarly to previous reports, the flow cytometry analysis revealed that the YhFF cell lines displayed mesenchymal stromal cell characteristics. Conclusion: This study will contribute to the development of clinical-grade cell-based products such as micro-vesicles and exosomes for future therapeutic applications in regenerative medicine. Key words: Cell therapy, Fibroblasts, Mesenchymal stem/stromal cells, Human embryonic stem cells, Micro-vesicles

Recent microfluidic innovations for sperm sorting

Sperm selection is a clinical need for guided fertilization in men with low-quality semen. In this regard, microfluidics can provide an enabling platform for the precise manipulation and separation of high-quality sperm cells through applying various stimuli, including chemical agents, mechanical forces, and thermal gradients. In addition, microfluidic platforms can help to guide sperms and oocytes for controlled in vitro fertilization or sperm sorting using both passive and active methods. Herein, we present a detailed review of the use of various microfluidic methods for sorting and categorizing sperms for different applications. The advantages and disadvantages of each method are further discussed and future perspectives in the field are given

Xeno-free generation of new Yazd human embryonic stem cell lines (Yazd4-7) as a prior stage toward good manufacturing practice of clinical-grade raw materials from discarded embryos: A lab resources report

Background: Xeno-free generation of human embryonic stem cells (hESCs) is important to prevent potential animal contaminations in culture for advanced cellbased therapeutic applications. Xeno-free production of hESCs is the first step for manufacturing clinical-grade hESC lines. Objective: To produce new hESC lines in xeno-free condition. Materials and Methods: This lab resources report was conducted at Stem Cell Biology Research Center, Yazd, Iran from 2019-2022. 4 new hESC lines from 11 (10 fresh and 1 frozen) donated surplus discarded human embryos were established. In this study, we report the xeno-free derivation of new Yazd hESC lines (Yazd4-7), without using immunosurgery, by culturing intact zona-free blastocysts obtained from discarded embryos onto the YhFF#8 cells as a feeder layer in a microdrop culture system. The pluripotency gene expression profile of the cell lines was assessed by reverse transcription polymerase chain reaction and the expression of specific surface markers was detected using immunofluorescent staining. In vitro differentiation was induced using embryoid body formation and gene expression profile of 3 germ layers and germ cells. Reverse transcriptase polymerase chain reaction was investigated to prove their pluripotent capacity. Results: In sum, we have been able to generate 4 new hESC lines (Yazd4-7) from 11 discarded embryos in xeno-free culture conditions using a micro drop culture system and YhFF#8 as a human source feeder layer. Conclusion: The outcome of this work can be the foundation for the future allogeneic cell-based therapeutic application using clinical grade good manufacturing practicederived hESC derivatives. Key words: Derivation, Human embryonic stem cells, Human foreskin fibroblasts, Xeno-free, Good manufacturing practice, Mouse embryonic fibroblasts

The effect of the human cumulus cells-conditioned medium on in vitro maturation of mouse oocyte: An experimental study

Background: To increase the results of infertility treatment, many efforts have been made to improve the treatment methods. As assisted reproductive technology is mainly using cell culture methods, one of the approaches to improve this technology is conditioned medium from different sources. It is desirable to apply in vitro maturation (IVM) and use oocytes from normal cycles instead of stimulating ovulation. Objective: To investigate the effect of human cumulus cell condition medium (hCCCM) on the IVM of immature mouse oocytes and morphology. Materials and Methods: In this experimental study, 240 germinal vesile oocytes were collected from four-six wk-old mice after 48 hr of 5IU pregnant mare serum gonadotropin (PMSG) injection and cultured in hCCCM (test group, n = 120) and DMEM + 20% FBS (control group, n = 120). The IVM rates and changes in perivitelline space (PVS) and shape were investigated at 8, 16, and 24 hr following the culture. The mature (MII) oocytes were subjected to in vitro fertilization (IVF) and the fertilization rate was assessed in three days. Results: A significant difference was observed between the maturation rates in the hCCCM and control groups (24.16% vs 0%; p = 0.001), as well as morphologic changes between the two groups (p = 0.04, p = 0.05). The development rate for MII oocytes attained from IVM in the hCCCM group was 27.58% (2-cell) and 6.89% (4-cell). Data displayed that hCCCM is an effective medium for oocytes maturation compared to the control medium. Conclusion: hCCCM supports oocyte in vitro growth and maturation. Moreover, hCCCM changes the oocyte shape and size of perivitelline space. Key words: Germinal vesicle, Cumulus cell, Conditioned medium, In vitro fertilization, In vitro maturation, Oocyte

Derivation of new human embryonic stem cell lines (Yazd1-3) and their vitrification using Cryotech and Cryowin tools: A lab resources report

Background: Cell banking initial outgrowths from newly derived human embryonic stem cells (hESCs) requires an efficient freezing method. Vitrification is used for the preservation of gametes and early embryos in assisted reproduction techniques (ART). Moreover, vitrification was applied for cryopreservation of hESCs using open pulled straws. Objective: To derive and characterize new hESC lines and then use Cryotech and Cryowin tools for their vitrification. Materials and Methods: Human ESC lines were generated in a microdrop culture system using mouse embryonic fibroblasts (MEFs) as the feeder layer; this was later scaled up using both MEFs and Yazd human foreskin fibroblasts batch 8 (YhFF#8). To bank the cell lines, master cell banks of 100 Cryotech and Cryowin tools were produced for each individual cell line using the vitrification method; flasks of hESC lines were also cryopreserved using a conventional slow-freezing method. Results: The pluripotency of cell lines was assessed by their expression of pluripotency-associated genes (OCT4/POU5F1, NANOG, and SOX2) and markers such as SSEA4, TRA-1-60, and TRA-2-49. Their in vitro capacity to differentiate into germ layers and germ cells using embryoid body (EB) formation and monolayer culture was assessed by screening the expression of differentiation-associated genes. The chromosomal constitution of each hESC line was assessed by G-banding karyotyping. Conclusion: Cryotech and Cryowin tools used to vitrify new hESCs at an early stage of derivation is an efficient means of preserving hESCs. Key words: Derivation, Human embryonic stem cells, Human foreskin fibroblast, Microdrop, Vitrification

The Effect of The Conditioned Medium from Human Embryonic Stem Cells on Mouse Oocytes In Vitro Maturation

Objective: Some reports have indicated that conditioned medium from growing mouse embryonic stem cells (ESCs)provides a supportive condition for small follicles growing, oocyte maturation, and following embryo growth. The aim ofthis study is assessing in vitro maturation (IVM) and consequent in vitro fertilization (IVF) outcome of immature mouseoocytes using human embryonic stem cells conditioned medium (HESCM).Materials and Methods: In this experimental study, 240 germinal vesicle (GV) oocytes were took from NMRI femalemice, aged 4-6 weeks, 48 hours before injection of 5 IU pregnant mare serum gonadotropin (PMSG). 120 GV oocyteswithout cumulus cells were cultured in each of the groups. 120 GV were cultured in HESCM as test groups and also120 GV cultured in human embryonic stem cells medium (HESM) as control groups. After evaluating the metaphase II(MII) oocyte maturation rate at 8, 16 and 24 hours, the MII oocytes subsequently were fertilized in vitro and the two-cellembryo development rate was recorded at days 1, 2, and 3. Statistical analysis was performed by using the generalizedestimating equations (GEE) method that calculated their rate ratio.Results: Our data indicated there are significant differences between the maturation rates in HESCM and HESM(P=0.004), also the two-cell embryo development was significant between two culture media (P=0.00).Conclusion: Similar to some other studies, the secretome of the HESCM showed a significant impact on the IVMoutcomes in mice

The stem cell session of the 7th Yazd International Congress and Student Award in Reproductive Medicine held at Shahid Sadoughi University of Medical Sciences

This paper summarizes the proceedings of the stem cell session of the “7th Yazd International Congress and Student Award in Reproductive Medicine” held at Shahid Sadoughi University of Medical Sciences, Yazd, Iran, on 28-30 April 2017. Here, we collected the papers of the session entitled: “Stem Cells, Good manufacturing practice, and tissue engineering”, that presented and discussed at this meeting by the international and national experts of the overlaps of the fields of stem cells and reproductive medicine, and the translation of these efforts towards practical application in regenerative medicine